The cutaneous lymphoid proliferations a comprehensive textbook of lymphocytic infiltrates of the ski
The Cutaneous Lymphoid Proliferations
A Comprehensive Textbook of Lymphocytic Infiltrates of the Skin 2nd
Edition Cynthia M. Magro
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Diagnosis of Cutaneous Lymphoid Infiltrates A Visual Approach to Differential Diagnosis and Knowledge Gaps Antonio Subtil
A Comprehensive Textbook of Lymphocytic Infiltrates of the Skin
Second Edition
Cynthia M. Magro MD
Professor of Pathology and Laboratory Medicine
Department of Pathology, Cornell University
Weill Cornell Medicine
New York, NY, USA
A. Neil Crowson MD
Clinical Professor of Dermatology, Pathology, and Surgery Director of Dermatopathology at the University of Oklahoma and Regional Medical Laboratory President of Pathology Laboratory Associates Tulsa, OK, USA
Martin C. Mihm MD
Clinical Professor of Pathology and Dermatology, Harvard Medical School Director of Melanoma Program, Dermatology, Brigham and Women’s Hospital Co-Director of Melanoma Program, Dana-Farber and Brigham and Women’s Cancer Center Director, Mihm Cutaneous Pathology Consultative Service Brigham and Women’s Hospital Boston, MA, USA
Published by John Wiley & Sons, Inc., Hoboken, New Jersey
Published simultaneously in Canada
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Contents
Acknowledgments, viii
1 Introduction to the Classification of Lymphoma, 1
Kiel Lukes–Collins, and Working Formulation classifications, 1
WHO, REAL, EORTC, and the Combined WHO/EORTC classifications, 2
Summary, 7 References, 8
Appendix: Definitions of key terms and techniques, 9
2 The Therapy of Cutaneous T Cell Lymphoma, 14
Benjamin H. Kaffenberger, Mark A. Bechtel, and Pierluigi Porcu
Lymphomatoid papulosis with a rearrangement of chromosome 6p25.3, 279
Cutaneous anaplastic large cell lymphoma, 280
Small cell ALCL, 282
Additional unusual histologic variants of anaplastic large cell lymphoma, 282
Breast-implant-associated anaplastic large cell lymphoma, 282
Intravascular anaplastic large cell lymphoma, 282
Sarcomatoid anaplastic large cell lymphoma (Case vignette 14), 283
CD30-positive large B cell lymphoma, 285
Case vignettes, 286 References, 309
14 CD4+ Peripheral T Cell Lymphoma, Not Otherwise
Specified, Including Primary Cutaneous Cd4+ Small/ Medium-Sized Pleomorphic T Cell Lymphoma, 312
Introduction, 312
Primary cutaneous CD4+ small/medium-sized pleomorphic T cell lymphoma, 312
CD30-negative large cell T cell lymphoma, 313
Cutaneous follicular helper T cell lymphoma, 314
Overview of overall prognosis of primary cutaneous peripheral T cell lymphoma, unspecified, 315
Evolution of the nomenclature of primary cutaneous CD4+ small/medium-sized pleomorphic T cell lymphoma, 319
Case vignettes, 320
References, 333
15 Subcutaneous Panniculitis-Like T Cell Lymphoma, 334
Clinical features, 334
Morphology, 336
Phenotype, 337
Molecular studies, 337
Differential diagnosis, 337
Case vignettes, 340
References, 349
16 CD8 T Cell Lymphoproliferative Disease of the Skin, 351
Overview, 351
Introduction, 351
Classification of primary CD8+ cutaneous T cell lymphomas, 352
Histomorphology of primary cutaneous CD8+ T cell lymphoma: primary cutaneous aggressive epidermotropic CD8+ T cell lymphoma, and CD8+ variants of peripheral T cell lymphoma, NOS, including primary
cutaneous CD8+ granulomatous T cell lymphoma, 353
CD8 variant of lymphomatoid papulosis and other related
CD30-positive T cell lymphoproliferative disorders of CD8 subtype, 354
Light microscopic findings, 354
Indolent CD8 positive lymphoid proliferation of the face and other body sites including acral surfaces, 355
CD8 prolymphocytic leukemia, 355
CD8 pseudolymphoma related to underlying HIV disease, 356
Drug-associated CD8+ pseudolymphoma, 356
Actinic reticuloid as a unique form of CD8+ pseudolymphoma, 356
Case vignettes, 357
References, 375
17 Nasal and Related Extranodal Natural Killer Cell/T Cell
Lymphomas and Blastic Plasmacytoid Dendritic Cell
Neoplasm, 377
Introduction, 377
Biology of NK and NK-like T cells, 377
NK/T-cell lymphoma, 379
Nasal NK/T cell lymphoma, 379
Nasal type NK/T cell lymphoma, 380
Aggressive NK cell lymphoma, 380
Role of Epstein–Barr virus in the evolution of NK/T cell lymphomas, 382
Blastic plasmacytoid dendritic cell neoplasm, 382
CD56-positive γ δ lymphoma involving the subcutaneous fat, 383
Hydroa vacciniforme-like EBV-associated T cell lymphoproliferative disease/mosquito bite hypersensitivity, 416
EBV+ cutaneous B cell lymphoproliferative disorder of the elderly, 420
EBV-associated mucocutaneous ulcer, 421
EBV + T cell lymphoproliferative disease of the elderly, 421
General principles regarding EBV-associated lymphomagenesis, 421
Pathogenetic link between EBV-associated B cell lymphoma and iatrogenic immune dysregulation related to either
methotrexate or cyclosporine, 421
Case vignettes, 423
References, 432
20 Hodgkin Lymphoma of the Skin, 435
Clinical features, 435
Subtypes of Hodgkin lymphoma, 436 References, 447
21 Chronic Lymphocytic Leukemia of B Cell and T Cell
Prolymphocytic Leukemia, 449
B cell chronic lymphocytic leukemia, 449
T cell prolymphocytic leukemia, 452
Case vignettes, 455 References, 471
22 Adult T Cell Leukemia/Lymphoma, 473
Clinical features, 473
Pathology, 474
Phenotypic studies, 475
Pathogenesis, 475
Infective dermatitis of childhood, 476
Case vignettes, 477
References, 484
23 Angioimmunoblastic Lymphadenopathy/
Angioimmunoblastic T Cell Lymphoma, 486
Clinical features, 486
Light microscopic findings, 487
Phenotypic studies, 488
Molecular studies, 488
Pathogenesis, 489
Case vignettes, 491
References, 497
24 Lymphomatoid Granulomatosis, 499
Introduction, 499
Clinical features, 499
Histopathology, 500
Histogenesis, 501
Clonality studies, 501
Differential diagnosis, 501
Treatment, 502
Case vignette, 503
References, 506
25 Cutaneous Infiltrates of Myeloid Derivation 507
Introduction, 507
Leukemia cutis, 507
Clonal histiocytopathy syndromes, 509
Histiocytopathy of factor XIIIA perivascular dermal dendritic cell origin, 514
Case vignettes, 517
References, 537
Index, 541
Acknowledgments
The authors would like to express special thanks for editorial support to Arthi Kumar at New York-Presbyterian/Queens Hospital and Shabnam Momtahen of Weill Cornell Medicine. Their assistance has been invaluable.
Cynthia M. Magro
A. Neil Crowson
Martin C. Mihm
Introduction to the Classification of Lymphoma
Kiel Lukes–Collins, and Working Formulation classifications
Since the first edition of this book, further clinical, morphologic and genetic research has continued to shed light on the different aspects of lymphoma. A significant revision of the classification was published in 2008 (Swerdlow et al., 2008), which highlights the extensive advances that have been made over the decades in understanding these hematologic disorders. The prominent aspects of this approach will be considered, as well as the basis for the new recommendations. A review of some suggestions for further classifications of T cell lymphomas will also be detailed. The earliest classification schemes were based on architectural criteria; specifically, lymphomas were categorized in terms of those that assumed a diffuse versus a nodular growth pattern (Rappaport et al., 1956; Lennert et al., 1975; Lennert, 1978; Lennert and Feller, 1992). In the 1960s, the Rappaport classification scheme, prior to the advent of immunophenotyping, added a consideration of the cell type. In that classification scheme, the large lymphocytes were, not surprisingly, mistaken for histiocytes. Thus, for example, that scheme recognized a diffuse histiocytic lymphoma, which we now know to derive from lymphocytes and to be, most often, a diffuse large B cell lymphoma. With the use of immunophenotyping, and the recognition of the distinction between T and B lymphocytes and histiocytes, new approaches to lymphoma classification emerged. One such scheme, designated the Kiel classification (see Table 1.1), graded lymphoid neoplasms into low-grade versus high-grade lymphomas and attempted to relate the cell types
B cell
Lymphocytic
identified in any particular lymphoma to their non-neoplastic counterparts in the benign lymph node (Gerard-Marchant et al., 1974; Lennert et al., 1975; Lennert, 1978, 1981; Stansfield et al., 1988; Lennert and Feller, 1992). Popular in the Western hemisphere from the mid-1970s to the mid-1980s, the Lukes–Collins classification emphasized immunophenotypic profiling (Lukes and Collins, 1974).
In the early 1980s, the International Working Formulation categorized lymphoid neoplasms into low, intermediate, and high grade malignancies based on clinical aggressiveness in concert with light microscopic findings. The goal was to produce a categorization of hematologic malignancies regardless of site of origin that was clinically useful, yet had scientific merit and diagnostic reproducibility (the non-Hodgkin pathological classification project 1982). Although the Kiel classification presaged the Working Formulation, this newer classification scheme did not emphasize B and/or T cell ontogeny per se; this was in contradistinction to the updated Kiel classification (Table 1.2). Among the low-grade malignancies were small lymphocytic lymphoma, chronic lymphocytic leukemia, small cleaved follicular lymphoma, and follicular lymphoma of mixed cell type. The intermediate-grade tumors included malignant lymphoma of follicle center cell origin with a predominance of large cells, diffuse lymphoma of small cleaved cells, and diffuse lymphoma of mixed and/or cleaved or noncleaved large cell type. The high-grade tumors were the diffuse immunoblastic, lymphoblastic, and Burkitt’s lymphoma. The cytomorphology
T cell
Low grade
Lymphocytic
Chronic lymphocytic and prolymphocytic leukemiaChronic lymphocytic and prolymphocytic leukemia
Source: Non-Hodgkin lymphoma pathologic classification project, 1982. Reproduced with permission of John Wiley & Sons.
and architecture were clearly of cardinal importance and, in essence, took precedence over the cell of origin in this classification scheme.
By the mid-1990s there was sufficient data gleaned from immunohistochemistry, cytogenetics, and molecular techniques to better categorize these tumors as distinct clinical and pathological entities manifesting reproducible phenotypic, cytogenetic, and molecular features, all defining critical determinants in the clinical course and prognosis. To attempt to evaluate whether a new classification scheme could be devised, a panel of 19 hematopathologists from Europe and the United States met to evaluate the current classification systems to consider whether a synthesis of the prior efforts could be made into a more usable and practical device to aid pathologists and clinicians. The classifications under consideration were the Kiel classification (Gerard-Marchant et al., 1974; Lennert et al., 1975; Lennert, 1978, 1981; Stansfield et al., 1988; Lennert and Feller, 1992), the Lukes–Collins classification (Lukes and Collins, 1974), and the Working Formulation (non-Hodgkin lymphoma pathologic classification project, 1982). What ultimately eventuated from this meeting was the Revised European–American Classification of Lymphoid Neoplasms (REAL classification) (see Table 1.3). It represented a synopsis of the existing hematologic literature, allowing categorization based on distinctive forms of hematopoietic and lymphoid malignancy separated on the basis of their peculiar clinical, light microscopic, phenotypic, molecular, and cytogenetic profiles (Harris et al., 1994; Cogliatti and Schmid, 2002).
B cell chronic lymphocytic leukemia/small lymphocytic lymphoma
B cell prolymphocytic leukemia
Lymphoplasmacytic lymphoma
Splenic marginal zone B cell lymphoma (+/−villous lymphocytes)
Hairy cell leukemia
Plasma cell myeloma/plasmacytoma
Extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid
tissue type
Nodal marginal zone lymphoma (+/−monocytoid B-cells)
Follicle center lymphoma, follicular,
Mantle cell lymphoma
Diffuse large cell B cell lymphoma
Mediastinal large B cell lymphoma
Primary effusion lymphoma
Burkitt’s lymphoma/Burkitt’s cell leukemia
T cell and natural killer cell neoplasms
Precursor T cell neoplasm
Precursor T lymphoblastic lymphoma/leukemia
Mature (peripheral) T cell and NK cell neoplasms
T cell prolymphocytic leukemia
T cell granular lymphocytic leukemia
Aggressive NK cell leukemia
Adult T cell lymphoma/leukemia (HTLV-1+)
Extranodal NK/T cell lymphoma, nasal type
Enteropathy-type T cell lymphoma
Hepatosplenic γ /δ T cell lymphoma
Mycosis fungoides/Sézary syndrome
Anaplastic large cell lymphoma, T/null cell, primary cutaneous type
Peripheral T cell lymphoma, not otherwise characterized
Angioimmunoblastic T cell lymphoma
Anaplastic large cell lymphoma, T/null cell, primary systemic type
Hodgkin lymphoma
Nodular lymphocyte predominance Hodgkin lymphoma
Classical Hodgkin lymphoma
Nodular sclerosis Hodgkin lymphoma
Lymphocyte-rich classical Hodgkin lymphoma
Mixed cellularity Hodgkin lymphoma
Lymphocyte depletion Hodgkin lymphoma
WhO, reaL, eOrtC, and the Combined WhO/ eOrtC classifications
The new WHO classification was a modest revision of the REAL classification, once again amalgamating reproducible clinical, light microscopic, phenotypic, molecular, and cytogenetic features into a coherent scheme (Jaffe et al., 2001; Cogliatti and Schmid, 2002). The concept of a classification scheme based purely on morphology was now considered archaic. However, the WHO/REAL classification was deficient from the perspective of cutaneous hematologic dyscrasias, as will be alluded to presently (Cogliatti and Schmid, 2002) (Table 1.3). Hence, in 1997 the European Organization for the Research and Treatment of Cancer (EORTC) established a scheme for the classification of cutaneous lymphomas (see Table 1.4). This classification scheme was met with criticism for reasons that will be discussed. Among the distinct clinical and pathological entities that were recognized by the EORTC classification were mycosis fungoides, including specific variants, lymphomatoid papulosis, large cell CD30-positive lymphoma, large cell CD30-negative lymphoma, panniculitis-like T cell lymphoma, marginal zone B cell lymphoma, primary cutaneous follicle center cell lymphoma, primary cutaneous large B cell lymphoma of the leg, and primary cutaneous plasmacytoma (Willemze et al., 1997) (Table 1.4). The main problem with this classification scheme was not the specific entities per se or even their purported clinical behavior. The difficulty
Source: Harris et al., 2000. Reproduced with permission of Oxford University Press.
Table 1.4 EORTC Classification for Primary Cutaneous Lymphomas
Source: Willemze et al., 1997. Reproduced with permission of American Society of Hematology.
was that there were a number of cutaneous hematologic dyscrasias that either were not included in this classification scheme or were phenotypically and biologically disparate, yet had to be forced into the same category. For example, both diffuse large B cell lymphomas of the trunk without features of follicle center cell origin and CD30-negative large cell T cell lymphoma would be categorized as CD30-negative large cell lymphomas. However, they are different from a prognostic perspective, the former being indolent and the latter being an aggressive form of lymphoma. Adult T cell leukemia lymphoma, nasal and extranodal NK/T cell lymphoma, nasal type, angioimmunoblastic T cell lymphoma, and T prolymphocytic leukemia commonly involve the skin as part of a disseminated lymphomatous process, yet they were not recognized in this classification scheme (Cogliatti and Schmid, 2002; Willemze et al., 2005).
Those who were proponents of the updated WHO classification (i.e., the REAL classification) contended that the WHO scheme was superior to the EORTC classification of cutaneous lymphomas. However, in the REAL/WHO classification scheme, there was only recognition of a few distinctive forms of cutaneous lymphoma, namely, mycosis fungoides, Sézary syndrome, and panniculitis-like T cell lymphoma. All of the other lymphomas were in the context of disease not specifically involving the skin, albeit recognizing that the diagnostic terms rendered could certainly be applied to various cutaneous lymphomas, including anaplastic large cell lymphoma, peripheral T cell lymphoma, not otherwise specified, NK/T cell lymphoma, extranodal marginal zone lymphoma, follicular lymphoma, diffuse large B cell lymphoma, and extramedullary plasmacytoma. Furthermore, all of the systemic and/or extracutaneous lymphomas that commonly involved the skin, such as adult T cell leukemia lymphoma were recognized by the WHO (Harris et al., 1994; Jaffe et al., 2001). Thus, the advantage of this classification scheme was that it encompassed a much broader spectrum of hematologic diseases having the potential to involve the skin. The problem was the radical difference in prognosis between the various lymphomas at extracutaneous sites relative to their behavior when presenting as
primary cutaneous neoplasms. Perhaps the best example of this is primary cutaneous follicle center lymphoma and primary cutaneous diffuse large cell B cell lymphoma, which can represent indolent forms of malignancy in the skin. The same potentially benign clinical course may apply to primary cutaneous anaplastic large cell lymphoma and localized peripheral T cell lymphoma in the skin, when dominated by small- and medium-sized lymphocytes.
To address the deficiencies in both the WHO and EORTC schemes as they apply to cutaneous hematologic disorders, a group of dermatologists and pathologists met in Lyon, France and Zurich, Switzerland in 2003 and 2004. The result was a publication that represents an amicable marriage, falling under the designation of the joint WHO–EORTC classification for cutaneous lymphomas (Jaffe et al., 2001; Cogliatti and Schmid, 2002; Burg et al., 2005; Slater, 2005; Willemze et al., 2005) (see Table 1.5). The WHO–EORTC classification recognizes 10 types of cutaneous T cell lymphoma and 4 forms of cutaneous B cell lymphoma, with clinical outcomes for those neoplasms designated as primary cutaneous lymphomas being recognized as distinct and separate from their extracutaneous counterparts. For example, diffuse large B cell lymphoma of follicle center cell origin is an indolent lymphoma while the “leg” type is an intermediate-prognosis lymphoma. The WHO–EORTC classification scheme also recognizes hematodermic neoplasm, which is a nonlymphoid tumor; hematodermic neoplasm now falls under the designation of blastic plasmacytoid dendritic cell neoplasm. Furthermore, it does include systemic lymphomas that commonly involve the skin, such as adult T cell leukemia lymphoma and intravascular large B cell lymphoma. The main deficiencies are the failure to include certain lymphoid neoplasms that characteristically involve the skin, namely, primary cutaneous B cell lymphoblastic lymphoma, angioimmunoblastic lymphadenopathy, lymphomatoid granulomatosis, and T cell prolymphocytic leukemia. In addition, while it does consider folliculotropic mycosis fungoides, there is no mention of syringotropic mycosis fungoides. The scheme does not address primary cutaneous post-transplant lymphoproliferative
Table 1.5 WHO–EORTC Classification of Cutaneous Lymphomas
Primary cutaneous CD4+ small/medium sized pleomorphic T cell lymphoma (provisional)
Cutaneous B cell lymphomas
Primary cutaneous marginal zone B cell lymphoma
Primary cutaneous follicle center lymphoma
Primary cutaneous diffuse large B cell lymphoma, leg type
Primary cutaneous diffuse large B cell lymphoma, other
Intravascular large B cell lymphoma
Precursor hematologic neoplasm
CD4+/CD56+ hematodermic neoplasm (blastic NK cell lymphoma)
Source: Willemze et al., 1997. Reproduced with permission of American Society of Hematology.
disease (PTLD) and methotrexate associated lymphoproliferative disease, although most of these in fact would fall in the category of diffuse large B cell lymphoma or anaplastic large cell lymphoma. As regards to PTLD, polymorphic variants and plasmacytic hyperplasia, however, would not be recognized. In contrast, the WHO considers these categories of iatrogenic dyscrasia (Jaffe et al., 2001). Other Epstein–Barr virus (EBV)-related disorders, such as plasmablastic lymphoma and hydroa vacciniforme-like lesions are not considered. It does not recognize those primary cutaneous small/medium sized pleomorphic T cell lymphomas that are rarely of the CD8 subset and which are to be distinguished prognostically from primary cutaneous aggressive epidermotropic CD8-positive T cell lymphoma. The designation of peripheral T cell lymphoma, type unspecified, can denote an aggressive form of cutaneous T cell lymphoma, however. The more accurate designation is that of CD30 negative large T cell lymphoma and one could argue that the latter designation would be more apposite. While the new scheme does consider hematodermic neoplasm a tumor of monocytic derivation, there is no consideration of granulocytic sarcoma, the histiocytopathies, or mast cell disease. The endogenous T cell dyscrasias that may presage lymphoma such as syringolymphoid hyperplasia with alopecia, atypical lymphocytic lobular panniculitis, pigmented purpuric dermatosis, and pityriasis lichenoides are not part of the classification scheme. Despite these deficiencies, it is to date the most accurate classification scheme for the categorization of hematologic diseases expressed in the skin (Burg et al., 2005; Willemze et al., 2005).
Since the 2006 WHO/EORTC classification of cutaneous lymphoma, further modifications have not been made of this classification scheme, although there are a number of emerging lymphoproliferative disorders, all of which we will consider in this latest edition of the book, including the new variants of lymphomatoid papulosis, indolent CD8 lymphoid proliferation, EBV+ lymphoproliferative disease of the elderly, indolent variants of gamma delta T cell lymphoma, and double-hit lymphoma. However, an important modification made by the International Society for Cutaneous Lymphoma/EORTC for the TNM classification of mycosis fungoides (MF) and Sézary syndrome was published in 2007 (Kim et al., 2007). It was the advancement in the understanding of the pathophysiology, including the cytogenetic and molecular basis of MF/SS that emerged as the impetus for the revised TNM classification of MF/SS presented in Table 1.6. The basic principles are identical to those outlined in the 1979 classification scheme. In the revised classification scheme, T0, as defined by lesions that are clinically and or histopathologically suspicious for MF/SS no longer exists. Another modification reflects the designated T1 and T2 subscript as “a” for cases that are exclusively in the context of patch stage MF and “b” for cases that manifest a patch/plaque stage overlap. For skin, patch indicates any size skin lesion without significant elevation or induration. Presence/absence of hypo- or hyperpigmentation, scale, crusting, and/or poikiloderma is noted. A plaque indicates any size skin lesion that is elevated or indurated. Presence or absence of scale, crusting, and/or poikiloderma is noted. The percentage of the skin involved is another important staging determinant. In the 1979 classification, it was assumed that the palm represented 1% of the body surface area; however, the revised updated classification scheme indicates that the palm represents approximately 0.5% of the body surface area. Another methodology for calculating percentage of body surface involved addresses the percentage of the skin involved in 12 specific regions and then tabulates the cumulative percentages. In the revised classification scheme, ulceration does not define a criterion for warranting
Table 1.6 ISCL/EORTC revision to the classification of mycosis fungoides and Sézary syndrome
TNMB stages
Skin
T1
T2
T3
T4
Node
N0
N1
N1a
N1b
N2
N2a
N3
N2b
Limited patches, papules, and/or plaques covering < 10% of the skin surface. May further stratify into T1a (patch only) versus T1b (plaque ± patch).
Patches, papules or plaques covering ≥ 10% of the skin surface. May further stratify into T2a (patch only) versus T2b (plaque ± patch).
One or more tumors (≥ 1-cm diameter)
Confluence of erythema covering ≥ 80% body surface area
No clinically abnormal peripheral lymph nodes; biopsy not required
Dutch grades 3-4 or NCI LN4; clone positive or negative
N x Clinically abnormal peripheral lymph nodes; no histologic confirmation
Visceral
M0 No visceral organ involvement
M1
Blood
B0
B0a
B0b
B1
B1a
B2
B1b
Visceral involvement (must have pathology confirmation and organ involved should be specified)
Absence of significant blood involvement: ≤ 5% of peripheral blood lymphocytes are atypical (Sézary) cells
Clone negative
Clone positive
Low blood tumor burden: > 5% of peripheral blood lymphocytes are atypical (Sézary) cells but does not meet the criteria of B2
Clone negative
Clone positive
High blood tumor burden: ≥ 1000/μL Sézary cells with positive clone
N, node; B, blood; T, tumor; M, metastatic; ISCL, International Society of Cutaneous Lymphoma; EORTC, European Organization for the Research and Treatment of Cancer.
the designation of tumor stage MF. To qualify as tumor stage MF requires at least one tumor 1.5 cm in diameter. The total number of lesions, total volume of lesions, largest size lesion, and region of body involved is documented. Erythroderma qualifies as T4, independent of whether or not the biopsy shows neoplastic T cell infiltration. They isolate only two histologic features of prognostic significance, namely variants of MF showing folliculotropism, which are classified as representing either a T1 or T2 form of the disease. The second histologic feature is one of large cell transformation, defined as a biopsy specimen showing large cells (≥ 4 times the size of a small lymphocyte) in 25% or more of the dermal infiltrate. The large cells are then evaluated for expression of CD30, given the prognostic significance of cases showing CD30-positive large cell transformation versus cases of large cell transformation that are CD30 negative. The lymph node alterations range from dermatopathic lymphadenitis (N1) and collections of atypical lymphocytes (N2), to one of frank effacement of the lymph node (N3). Atypical lymphocytes may be small (6–10 μm) or large (> 11.5 μm) cells; the cells exhibit irregularly folded, hyperconvoluted nuclei. In the revised ISCL/EORTC classification, clonality in the lymph node in the absence of any histologic abnormalities does not alter the staging. Abnormal peripheral lymph node(s) indicates any palpable
peripheral node that on physical examination is firm, irregular, clustered, fixed or 1.5 cm or larger in diameter. Node groups examined on physical examination include cervical, supraclavicular, epitrochlear, axillary, and inguinal. Central nodes, which are not generally amenable to pathologic assessment, are not currently considered in the nodal classification unless used to establish N3 histopathologically. Peripheral blood involvement has been recategorized whereby B0 represents 5% or less circulating Sézary cells, B2 is now defined as a clonal rearrangement of the TCR in the blood and either 1.0 K/μL or more Sézary cells or one of two phenotypic criteria being T cells with CD4/CD8 of 10 or more, or an increase in circulating CD4+ T cells that show a loss of CD7 or CD26 representing 40% or 30%, respectively, of the peripheral blood CD4 T cells. B1 is defined as more than 5% Sézary cells, but either less than 1.0 K/μL absolute Sézary cells or absence of a clonal rearrangement of the TCR, or both (Kim et al., 2007).
In addition, the International Society of Cutaneous Lymphoma and the EORTC created a risk stratification for cutaneous lymphoma other than MF and Sézary syndrome. In this risk stratification scheme, they proposed a TNM classification for non-MF/ SS cutaneous lymphomas, as summarized in Table 1.7. The authors emphasized the importance of a complete history/review of systems (e.g., +/− B-symptoms, organ-specific signs) and a thorough physical examination. Among the important laboratory values are a complete blood count with differential, and a comprehensive blood chemistry measurement, including lactate dehydrogenase (LDH). They recommend appropriate imaging studies, including the neck for evaluation of the cervical lymph nodes in cases showing significant head and neck involvement. Biopsies of suspicious extracutaneous sites are encouraged. They also suggest a bone marrow biopsy and aspirate should be performed in patients at risk of marrow involvement, especially in more aggressive forms of lymphoma, such as natural killer (NK)/T cell, aggressive CD8 + T cell and γ/δ T cell lymphoma and diffuse large B cell lymphoma, leg type). A bone marrow is not required in cases of indolent lymphoproliferative disease. A negative marrow involvement would further confirm that the skin involvement is primary and not secondary to a primary extracutaneous presentation. A lumbar puncture and spinal fluid
T
T1: Solitary skin involvement
T1a: a solitary lesion ≤5 cm diameter
T1b: a solitary >5 cm diameter
T2: Regional skin involvement: multiple lesions limited to one body region or two contiguous body regions
T2a: all disease encompassing in a ≤15-cm-diameter circular area
T2b: all disease encompassing in a >15 ≤30-cm-diameter circular area
T2c: all disease encompassing in a >30-cm-diameter circular area
T3: Generalized skin involvement
N
T3a: multiple lesions involving two noncontiguous body regions
T3b: multiple lesions involving at least three body regions
N0: No clinical or pathologic lymph node involvement
N1: Involvement of one peripheral lymph node region that drains an area of current or prior skin involvement
N2: Involvement of two or more peripheral lymph node regions or involvement of any lymph node region that does not drain an area of current or prior skin involvement
N3: Involvement of central lymph nodes
M
M0: No evidence of extracutaneous non-lymph node disease
M1: Extracutaneous non-lymph node disease present
MF, mycosis fungoides; SS, Sezary syndrome; T, tumor; N, node; M, metastatic.
assessment is recommended for patients with NK/T cell lymphoma (Kim et al., 2007). A bone biopsy is recommended for all cases of diffuse large B cell lymphoma of leg type. Some physicians suggest a bone marrow assessment in cases of primary cutaneous follicle center lymphoma because of the reported incidence of bone marrow involvement in 10% of cases, which in turn is associated with an inferior survival. The international extranodal lymphoma study group emphasize three clinical parameters that are of prognostic value, namely elevated LDH, the presence of two or more lesions, and a cutaneous tumor that manifests a nodular morphology in the setting of primary cutaneous marginal zone lymphoma and primary cutaneous follicle center lymphoma.(Senff et al., 2008)
The frequency and the clinical pathological spectrum of lymphomas of the skin diagnosed between the years of 2006 and 2013 at a major referral center in Austria, as categorized according to the two main recent classification schemes, namely the WHO/EORTC and the TNM ISCL/EORTC classifications, was recently published in 2015. Eighty-three percent of their cases fell into the cutaneous T cell lymphoma category with 60% of these cases being represented by mycosis fungoides, followed in decreasing order by CD-30positive lymphoproliferative disease, primary cutaneous CD4+ small/ medium-sized pleomorphic T cell lymphoma, Sézary syndrome and subcutaneous panniculitis-like T cell lymphoma. Not surprisingly, the most common B cell lymphomas were marginal zone lymphoma, primary cutaneous follicle center lymphoma and diffuse large B cell lymphoma of leg type. Their experience in terms of disease frequency, clinical features, and prognosis mirrors most major academic centers. In their study they also found a male predominance, an increasing incidence of cutaneous lymphoma incidence with age, and a greater age of onset of B cell lymphoma in women compared to men (Eder et al., 2015).
While there have not been any further updates of the 2006 EORTCWHO classification of cutaneous lymphoma, the 4th edition of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues was published in 2008 by the International Agency for Research on Cancer (Swerdlow et al., 2008; Jaffe et al., 2009; Campo et al., 2011). It was a modification of the earlier WHO classification of hematologic disorders based on the exact same philosophy as that which formulated the earlier WHO classification. In particular, hematologic disorders were considered as distinct clinicopathological entities where the combination of the clinical features, morphology, phenotypic profile, molecular features, and cytogenetics defined the entity with a precision that reflects the striking advances in our understanding of the genetic and epigenetic basis of disease. Compared to the earlier WHO classification of lymphoma, a far greater number of primary cutaneous lymphomas were recognized.
In the category of mature B cell neoplasms, the two primary cutaneous forms of B cell lymphoma that are recognized in the new 2008 classification of hematologic dyscrasias are diffuse large b cell lymphoma of leg type and primary cutaneous follicle center lymphoma. In the category of mature T and NK cell neoplasms, mycosis fungoides, Sézary syndrome, primary cutaneous gamma delta T cell lymphoma, primary cutaneous CD30-positive T cell lymphoproliferative disease, primary cutaneous CD4+ small/medium sized pleomorphic T cell lymphoma and subcutaneous panniculitis-like T cell lymphoma are described. The variants of mycosis fungoides recognized include follicular MF, pagetoid reticulosis, and granulomatous slack skin. Each lymphoma is presented as a distinct clinical pathological entity with unique clinical and histologic features, a distinctive phenotypic, molecular and cytogenetic, and oncogenic gene profile. The evolution of the current classification to one of precision at the exact
Table 1.7 TNM Classification for lymphomas other than MF and SS
Table 1.8 The 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues
MYELOPROLIFERATIVE NEOPLASMS
Chronic myelogenous leukaemia, BCR-ABL 1 positive
Chronic neutrophilic leukaemia
Polycythaemia vera
Primary myelofibrosis
Essential thrombocythaemia
Chronic eosinophilic leukaemia, NOS
Mastocytosis
Cutaneous mastocytosis
Systemic mastocytosis
Mast cell leukaemia
Mast cell sarcoma
Extracutaneous mastocytoma
Myeloproliferative neoplasm, unclassifiable
MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA AND ABNORMALITIES OF PDGFRA, PDGFRB OR FGFR1
Myeloid and lymphoid neoplasms with PDGFRA rearrangement
Myeloid neoplasms with PDGFRB rearrangement
Myeloid and lymphoid neoplasms with FGFR1 abnormalities
The italicized histologic types are provisional entities, for which the WHO Working Group felt there was insufficient evidence to recognize as distinct diseases at this time.
*These lesions are classified according to the leukaemia or lymphomas to which they correspond, and are assigned the respective ICD-O code.
genomic level is a dichotomous contrast to the original nascent classification scheme, which recognized only cell size and architecture. A summary of the classification scheme is presented in Table 1.8.
Summary
Tables 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 1.7 summarize the classification schemes as they have evolved over time. It should be apparent to the reader that the most recent classification scheme is certainly apropos, but still not globally inclusive. Each of the conditions listed in the classification scheme are discussed in the ensuing chapters, emphasizing the approach that should be given to each hematologic dyscrasia. Specifically, the entities are presented in the context of an integration of clinical, light microscopic, phenotypic, molecular, and cytogenetic data, and, where appropriate, additional considerations are given regarding pathobiology. Each cutaneous disorder truly has its own fingerprint; in this regard we have considered many of the individual hematologic disorders in their own respective chapters and/or considered no more than a few entities in a given chapter to emphasize the truly distinctive nature of so many of these disorders. In addition, we consider other forms of lymphoid dyscrasia that commonly involve the skin, recognizing that they are rare conditions and are still not part of the WHO–EORTC classification scheme.
references
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Cogliatti SB, Schmid U. Who is WHO and what was REAL? A review of the new WHO classification (2001) for malignant lymphomas. Swiss Med Wkly. 2002; 132:607–617.
Campo E1, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. Blood. 2011 May 12; 117(19):5019–5032.
Eder J, Kern A, Moser J, Kitzwögerer M, Sedivy R, Trautinger F. Frequency of primary cutaneous lymphoma variants in Austria: retrospective data from a dermatology referral centre between 2006 and 2013. J Eur Acad Dermatol Venereol. 2015 Jan 20. doi: 10.1111/jdv.12907. [Epub ahead of print] PubMed PMID: 25600184.
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Swerdlow SH, Campo E, Harris NL, et al WHO Classification of Tumours of Haematopoietic and Lymhoid Tissues, 4th edn. Lyon, France: IARC Press; 2008.
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appendix: Definitions of key terms and techniques
t cell antibodies
CD1a (T6, Leu6, OKT6, O10): An immature T cell antigen, found on cortical thymocytes and Langerhans cells, but not mature T cells.
CD2 (T11, Leu5, OKT11, MT910): A pan T cell antigen that corresponds to the sheep erythrocyte rosette receptor. It is present on all normal mature T cells.
CD3 (Leu4, T3, OKT3,SP7, PS1, Polyclonal): A pan-T cell antigen that is composed of five polypeptide chains covalently linked to the T cell receptor. All elements of the CD3/T cell receptor must be present for cell surface expression. Most anti-CD3 antibodies are directed toward the epsilon chain of the CD3/T cell receptor complex. The majority of mature T cells are CD3 positive. The CD3 antigen is first expressed in the cell cytoplasm and then on the surface. NK cells will manifest only cytoplasmic expression.
TCR-1, BF-1: They are antibodies that recognize the α/β heterodimer of the human T cell antigen receptor. It is expressed on normal mature peripheral blood T lymphocytes and on 50–70% of cortical thymocytes. The vast majority of T cell malignancies are derived from T cells of the αβ subtype.
TCR-gamma 1: An antibody that recognizes the γ/δ heterodimer portion of human T cell antigen receptor. It is present on a minor subset of CD3-positive T cells in peripheral blood, thymus, spleen, and lymph node.
CD5 (T1, Leu1, OKT1, CD5/54/F6, 4C7): A pan T cell antigen present on the majority of thymocytes and mature peripheral blood T cells; a loss of CD5 expression in T cells is indicative of ensuing neoplasia. The CD5 antigen is present on a small subset of normal B cells representing naïve B cells with endogenous autoreactive features and which have been implicated in innate immunity. It is also expressed on neoplastic B cell lymphoma cells of chronic lymphocytic leukemia, small lymphocytic lymphoma, rare cases of marginal zone lymphoma, and mantle zone lymphoma.
CD43 (DF-T1): This T-cell-associated antigen is expressed by normal T cells, granulocytes, and a subset of plasma cells, but not normal B cells. CD43 expression by a B cell is a feature of B cell neoplasia. Primary cutaneous diffuse large B cell lymphomas, marginal zone lymphomas, and follicle center cell lymphomas can be CD43-positive.
CD7 (Leu9, DK24): A pan T cell marker that is expressed by the majority of peripheral T cells. The expression of CD7 is an event that occurs relatively early in T cell ontogeny prior to rearrangement of the TCR-β chain. The CD7 antigen is expressed by both mature and immature T cell neoplasms. The CD7 antigen may not be expressed by memory T cells manifesting selective homing to the skin. Although a substantial reduction of this marker is characteristic for mycosis fungoides can be seen in other forms of peripheral T cell lymphoma, it is also diminished in the prelymphomatous T cell dyscrasias and many reactive dermatoses, albeit to a lesser degree than in mycosis fungoides. There is variation in the intensity of staining based on the detection system.
CD62L (LECAM-1, LAM-1, MEL-14): CD62L is part of the family of selectins that comprises three subcategories: L-selectin, E-selectin, and P-selectin designated as CD62L, CD62E, and CD62P, respectively. All of the selectins exhibit a similar glycan contributing to their adhesion function and participating in the interactions between inflammatory cells and endothelium. CD62L is expressed on blood monocytes, blood neutrophils, subsets of natural killer cells, and T and B lymphocytes,
including those of näıve phenotype. Virgin T cells in human peripheral blood uniformly express CD62L, whereas among the memory/effector population, the three predominant subsets are CD62L+/CLA+, CD62L+/CLA−, and CD62L−/CLA−.
CD4 (Leu3a, OKT4, MT310): A helper/inducer cell antigen. It is expressed by the majority of peripheral blood T cells and 80–90% of cortical thymocytes. Cortical thymocytes that are CD4-positive usually coexpress CD8. The majority of T cell neoplasms are of the CD4 subset. γδ T cells and NK cells are CD4negative. CD4 is also expressed by monocytes including, in the context of histiocytic proliferative disorders, myelomonocytic dyscrasias and hematodermic neoplasm.
CD8 (Leu 2a, C8/144B): A suppressor/cytotoxic cell antigen. The CD8 antigen is a 32 kilodalton heterodimeric protein that is expressed by approximately 30% of peripheral blood mononuclear cells and 60–85% of cortical thymocytes (P/F). Cortical thymocytes coexpress CD4. γδ Cells are frequently CD8-negative. A small percentage of peripheral T cell lymphomas are of the CD8 subset, such as primary cutaneous CD8-positive epidermotropic cytotoxic T cell lymphoma, some γδ T cell lymphomas, and panniculitis-like T cell lymphoma. Rarely, classic lesions of cutaneous T cell lymphoma (i.e., mycosis fungoides) will be CD8-positive. CD8 cells may be suppressive or cytotoxic in nature. The latter express cytotoxic proteins such as TIA and granzyme.
CD26: The protein encoded by the DPP4 gene is an antigenic enzyme expressed on the surface of most cell types and is associated with immune regulation, signal transduction and apoptosis. It is an intrinsic membrane glycoprotein and a serine exopeptidase that cleaves X-proline dipeptides from the N-terminus of polypeptides. The neoplastic cells of Sézary syndrome do not express CD26 and hence this particular marker is of value in the assessment of the peripheral blood in patients who are suspected as having Sézary syndrome.
CD52 (VTH34.5, Campath-1G): Expressed in lymphocytes, monocytes, eosinophils, thymocytes, and macrophages. It is expressed on most B and T cell lymphoid-derived malignancies; expression on myeloma cells is variable.
Cutaneous Lymphocyte Antigen (HECA-452): Expressed in memory T lymphocytes with preferential homing proportion to the skin endothelial cells and epithelial cells.
Fox P3 (236A/F7): Constitutive high expression of FOXP3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of FOXP3 in CD4+CD25− cells imparts a Treg phenotype in these cells.
TCL1 oncogene: The TCL1 locus on the chromosome 14q32.1 is associated with the development of leukemia when there is a translocation and or an inversion resulting in juxtaposition to various regulating elements of the T cell receptor. Tcl1 positivity is observed amidst the neoplastic cells in blastic plasmacytoid dendritic cell neoplasm, adult T cell leukemia, and T cell prolymphocytic leukemia.
NFATc : Calcineurin/Nuclear factor of activated T cells (NFAT) signaling plays a critical role in peripheral T-cell activation following TCR engagement. In resting cells, inactive NFAT transcription factors are located in the cytoplasm. Pathway activation leads to NFAT dephosphorylation, nuclear translocation, and activation of its transcriptional targets. In reactive lymphocytic infiltrates and early lesions of mycosis fungoides, the expression of NFAT is primarily confined to the cytoplasm. With advanced mycosis fungoides and or other forms of cutaneous T cell lymphoma, such as peripheral T cell lymphoma, type unspecified, there is acquisition of nuclear expression of NFAT within the nucleus. Of particular relevance is the finding that the catalytic domain of PLCG1 is frequently
mutated in tumoral samples of cutaneous T cell lymphoma and is associated with the nuclear expression of NFAT.
PD-1: Programmed death-1 (PD-1/CD279) cell surface protein, an inhibitory member of the CD28 costimulatory receptor superfamily, is expressed mainly in the subset of B cells, NK T cells, activated monocytes dendritic cells, activated T lymphocytes, and follicular helper T cells. The PD-1 pathway exerts its function through inhibiting TCR-mediated T cell proliferation and cytokine secretion, via its two ligands PD-L1 (B7-HICD274), and PD-L2 (B7-DC/CD273). PD1 is expressed in certain T cell malignancies of putative follicular helper T cell origin, including angioimmunoblastic lymphoma, primary cutaneous CD4+ small/medium-sized pleomorphic T cell lymphoma, and peripheral T cell lymphoma with a follicular pattern. In addition, in Sézary syndrome, the neoplastic cell populace is characteristically PD1 positive.
TOX: Thymocyte selection-associated high-mobility group box factor (TOX) is another critical regulator of early T-cell development, specifically during the transition from CD4+ CD8+ precursors to CD4+ T cells. However, upon completion of this process, it is tightly suppressed and mature CD4+ cells do not have significant TOX expression, except follicular helper T cells. There is significant upregulation of nuclear TOX expression in the neoplastic epidermotropic T cells of mycosis fungoides. Nuclear expression of TOX is not an absolute criterion of malignancy as it can be seen in reactive lymphocytes, although the extent and intensity of intraepidermal and dermal nuclear TOX expression amidst T cells is less in reactive inflammatory dermatoses. Since TOX is upregulated in follicular helper T cells, it is common to see very strong expression of TOX in cases of primary cutaneous CD4+ small/ medium-sized pleomorphic T-cell lymphoma.
plasma cell markers
CD138 (MI15): CD138/syndecan-1 protein backbone is a single chain molecule of 30.5 kDa. Five putative GAG attachment sites exist in the extracellular domain. GAG fine structure appears to reflect the cellular source of the syndecan. Expression of CD138 in human hematopoietic cells is restricted to plasma cells in normal bone marrow. Early B cell precursors in human bone marrow are CD138 negative. CD138 is also expressed in endothelial cells, fibroblasts, keratinocytes, and normal hepatocytes.
Natural killer cell-associated markers
CD16 (DJ130c): A natural killer cell and myelomonocytic antigen. It is expressed by all resting natural killer cells, neutrophils, and activated macrophages. It is also the antibody receptor for antibody dependent cellular cytotoxicity.
CD56 (MOC1, T199, C5.9): A natural killer cell antigen. This antigen is expressed by all resting and activated natural killer cells, a subset of cytotoxic T cells that mediates non-major histocompatibility complex (non-MHC) restricted cytotoxicity, and dendritic monocytes. However, it is expressed by other cell types including CD T cells and plasmacytoid dendritic cells, and myeloid leukemic cells can express CD56.
Cytotoxic protein markers
TIA
Perforin
Granzyme
B cell markers
The immunoglobulin light chains are the most reliable way of distinguishing a malignant B cell process from a reactive one (restricted light chain expression).
CD10 (CALLA): This B cell antigen was originally thought to be a tumor-specific marker expressed by neoplastic cells of acute lymphoblastic leukemia. The CD10 antigen can be expressed by follicular lymphomas, B cell lymphoblastic lymphomas, normal T cells undergoing apoptosis and certain T cell malignancies namely in the context of angioimmunoblastic lymphadenopathy.
CD19 (HD37): The CD19 antigen is expressed initially at the time of immunoglobulin heavy chain gene rearrangement. Anti-CD19 antibodies stain almost all cases of non-T cell acute lymphoblastic leukemia, as well as mature B cell leukemias and lymphomas. Restricted to use in flow cytometry or frozen tissues.
CD20 (B1, L26, Leu16): A pan B cell antigen that is expressed at the time of light chain gene rearrangement. Anti-CD20 antibodies react with 50% of immature B cell lymphoblastic leukemia cells. CD20 is not expressed by plasma cells. It can occasionally be expressed by neoplastic T cells and there is also a population of normal T cells that weakly expresses CD20.
CD22 (4 KB128, To15): A pan B cell antigen that is very similar to the CD20 antigen.
Bcl-1: Bcl-1/cyclin D1 belongs to the G1 cyclins and plays a key role in cell cycle regulation during the G1/S transition by cooperating with cyclin-dependent kinases (CDKs). Its overexpression may lead to growth advantage for tumor cells by way of enhanced cell cycle progression, and it has been reported in various human cancers, for example, esophageal, breast, and bladder carcinomas. Among hematolymphoid malignancies, cyclin D1 overexpression resulting from translocational activation has also been recognized in a subset of B chronic lymphocytic leukemia (BCLL), multiple myeloma, splenic marginal zone lymphoma, hairy cell leukemia, and mantle cell lymphoma.
Bcl-2: The bcl-2 family of proteins (bcl-2, bcl-w, bcl-xL, bcl-2 related protein A1, etc.) regulates outer mitochondrial membrane permeability. Bcl-2, bcl-w, bcl-xL, and bcl-2 related protein A1 are antiapoptotic members that prevent release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Bcl-2 and bcl-xL are present on the outer mitochondrial membrane and are also found on other membranes in some cell types. Bcl-w is required for normal sperm maturation. In the context of its value in lymphoid infiltrates, it is ubiquitously expressed by small mature lymphocytes. Normal germinal center cells are bcl-2 negative. In contrast, neoplastic germinal center cells can be bcl-2 positive and are typically positive in nodal follicular lymphoma. In primary cutaneous diffuse large cell lymphomas, bcl-2 expression is an adverse prognostic variable.
Bcl-6: Bcl-6 protein is expressed in B cell lymphomas of follicle center B cell origin.
Bcl-10: Apoptosis regulator B cell lymphoma 10 (bcl-10) may show aberrant nuclear expression in primary cutaneous marginal zone lymphomas associated with extracutaneous dissemination.
CD79a: CD79a is expressed during all phases of B cell ontogeny and in this regard, CD79a is positive in B cells in both early- and late-stage B cell ontogeny. It is expressed prior to the expression of CD20 and is retained in the postgerminal B cell after CD20 is no longer expressed. CD79a is involved in B cell receptor development whereby a genetic deletion of CD79a can prevent and halt B cell development. Since CD79a is expressed at all stages of B cell ontogeny, it is a valuable marker in concert with CD20; a decrement in the expression of CD79a would potentially signify B cell neoplasia.
PAX5: The PAX5 gene is a transcription factor that exhibits a highly conserved DNA binding motif that defines an important factor in the early development of B cells. It has been postulated that dysregulation of the PAX5 gene contributes to lymphomagenesis. It is expressed in mature B cells including Hodgkin lymphoma. There are rare cases of its expression in anaplastic large cell lymphoma.
Myelomonocytic markers including dendritic cell markers
CD15 (C3D-1): It is normally expressed on neutrophils and most forms of nonlymphoid acute leukemia. It is aberrantly expressed by Reed–Sternberg cells of Hodgkin lymphoma along with chronic lymphocytic leukemia and lymphoblastic lymphoma.
CD68 (PGM1, KP1): This antigen is found on monocytes, granulocytes, mast cells, and macrophages.
CD34 (QBEnd10): The CD34 antigen is a single-chain transmembrane glycoprotein that is associated with human hematopoietic progenitor cells. It is present on immature hematopoietic precursor cells and TdT-positive B cells and T lymphoid precursors. CD34 expression decreases as these hematopoietic precursors undergo progressive maturation. CD34 myeloid progenitors can differentiate into two major myeloid subsets in the skin: Langerhans cells and dermal interstitial dendrocytes. While these mature antigen-presenting cells are CD34 negative, the dermal dendritic and Langerhans cell precursors manifest a CD34+ CD14+ CD116+ phenotype. The quantity of CD34+ progenitor cells in the marrow is closely associated with advancement of disease in patients with chronic idiopathic myelofibrosis. Expectedly, patients with myelofibrosis can develop paraneoplastic Sweet’s-like reactions whereby the presence of CD34+ cells in the infiltrate could be a harbinger of a more accelerated clinical course (personal observations). CD34+ hematopoietic stem cells are the source of dermal fibrocytes involved in wound healing and representing the implicated fibrogenic cell of nephrogenic systemic fibrosis.
CD43: CD43 antigen is expressed by T cell lymphomas and about 30% of B cell lymphomas. CD43 is expressed on the membrane and in the cytoplasm of T cells and cells of myeloid lineage, including monocytes. CD43 expression by a B cell is a phenotypic aberration indicative of B cell neoplasia.
CD123: The protein encoded by this gene is an interleukin-3 (IL-3)-specific subunit of a heterodimeric cytokine receptor. The receptor is composed of a ligand-specific α subunit and a signal transducing β subunit shared by the receptors for IL-3, colony stimulating factor 2 (CSF2/GM-CSF), and interleukin-5 (IL-5). The binding of this protein to IL3 depends on the β subunit. The β subunit is activated by the ligand binding and is required for the biological activities of IL-3. This gene and the gene encoding the colony-stimulating factor 2 receptor α chain (CSF2RA) form a cytokine receptor gene cluster in an X–Y pseudoautosomal region on chromosomes X or Y. It is positive in acute myelogenous leukemia and blastic plasmacytoid dendritic cell tumor.
CD83: This protein is a member of the Ig superfamily manifesting expression on mature dendritic cells of all types, including plasmacytoid dendritic cells and Langerhans cells.
CD11c: CD11c transmembrane protein expressed at high levels on dendritic cells and monocytes that are likely destined to become dendritic cells. It is also positive on hairy cell leukemia cells and chronic lymphocytic leukemia cells.
MXA: MXA is a surrogate marker for the type-I-rich microenvironment. It is expressed in plasmacytoid dendritic cells and
hence can be expressed in neoplastic cells of the blastic plasmacytoid dendritic cell tumor. In addition, myeloid dendritic cells can express MXA. including in the context of a neoplastic counterpart characteristic of clonal myeloid dendritic cell dyscrasia, a marker of chronic myeloproliferative disease (i.e. myelofibrosis, chronic myelodysplastic syndrome, myelomonocytic leukemia)
Lysozyme: Lysozyme is also referred to as muramidase. It is a hydrolytic glycosidase with potential antibacterial properties. It is found in high concentrations in various bodily secretions and is present at high levels in egg whites. Lysozyme is expressed in macrophages and neutrophils. It is also expressed by earlier precursor cells of myelomonocytic derivation and hence is positive in myeloid, monocytic and myelomonocytic acute leukemias.
CD163: CD163 is a scavenger receptor for the hemoglobin haptoglobin complex and is expressed in macrophages. Certain terminally differentiated monocytes with dendritic cell properties may not be positive for CD163; for example, Langerhans cells do not express CD163. Acute myeloid leukemia with monocytic differentiation can, however, exhibit positivity for CD163.
Langerin: Langerin is a transmembrane receptor specific for Langerhans cells, manifesting localization to the Birbeck granule, where it plays a role in the internalization of antigen prior to antigen presentation to T cells. It is not expressed on indeterminate cells en route to the lymph node, but rather is expressed on immature Langerhans cells, which reside in the epidermis.
CD14. This molecule functions as a toll receptor and is a marker of terminally differentiated monocytes that are likely destined to become dendritic cells. It performs a critical function in the detection of bacterial lipopolysaccharide. While the dominant expression is by macrophages and other related mature monocytes, there is weak expression amidst neutrophils. The differentiation of the CD14 positive monocyte into a myeloid dendritic cell and other dendritic cell types occurs in the setting of a cytokine milieu rich in interleukin 4 and granlocyte macrophage colony-stimulating factor.
CD117: Mast/stem cell growth factor receptor(SCFR), also known asproto-oncogene c-CD117 falls under the alternative designations of tyrosine protein kinase and is a receptor tyrosine kinase protein that is encoded by the KIT gene. It is expressed in mast cells and in melanocytes, but it is also expressed by hematopoietic stem cell precursors. This latter cell type is normally present at very low levels in the peripheral blood; however, certain agents, such as granulocyte colonystimulating factor can lead to mobilization to the peripheral blood and extramedullary organ sites. CD117 is a proto-oncogene that is overexpressed in myeloid leukemias and of course is extensively positive in benign and neoplastic mast cell infiltrates.
Myeloperoxidase: Myeloperoxidase is a peroxidase enzyme that is abundantly expressed in neutrophils at high levels. Over and above its expression in mature granulocytes, is its positivity in neutrophil precursors. In this regard it is expressed in the setting of myeloid leukemia. Myeloperoxidase is also expressed in activated macrophages and therefore can be found in certain histiocyte-rich inflammatory conditions, such as Kikuchi’s disease, and in the setting of histiocytoid Sweet’s syndrome.
Follicular dendritic cell markers
CD21: CD21 also falls under the designation of the C3d receptor and Epstein Barr virus receptor. It is expressed on all mature B cells and follicular dendritic cells. It forms a complex with CD19 and CD81 defining the coreceptor B complex. It interacts with antigen and
optimizes the B cell response to antigen. CD21 is of value in the assessment of the follicular dendritic network in B cell proliferations, as significant disruption of the orderly follicular dendritic network in a germinal center is a feature of follicle center lymphoma and marginal zone lymphoma.
CD23: While there is no literature precedent on either the expression of CD23 in lesions of primary cutaneous B cell lymphoma, CD23 expression in non-neoplastic lymphoid cells is well described, occurring in naïve B cells, monocytes and follicular dendritic cells. In human tonsillar tissue, CD23 is a precentroblast marker; it is expressed on naïve B cells both in the mantle zone and early germinal center phase. It is upregulated in the early stages of B cell activation by interleukin 4 and functions as an IgE receptor and lymphocyte growth factor. CD23 also plays a role in the augmentation of B cell proliferation and of antigen presentation. Human B lymphocytes induced from a resting state to one of blastic transformation demonstrate CD23 expression.
CD35: CD35 also falls under the designation of Complement receptor type 1 (CR1) representing a glycoprotein found on erythrocytes, leukocytes, glomerular podocytes, hyalocytes, and splenic follicular dendritic cells. The protein is important in the mediation of interactions between effector cells and immune complexes containing activated complement. It plays a critical role in the removal of complement opsonized immune complexes. It is a negative regulator of the complement cascade, resulting in inhibition of both the classic and alternative pathways.
activation/proliferation markers
CD25 (Tac, ACT-1): An activation marker that detects the α chain of the interleukin-2 receptor. The CD25 antigen is a 55 kilodalton glycoprotein that is expressed by activated B and T lymphocytes and weakly by histiocytes. The CD25 antigen is strongly expressed by cutaneous T cell neoplasms undergoing transformation. The CD25 antigen is also expressed by the Reed–Sternberg cells of Hodgkin lymphoma.
CD30 (Ber-H2, Ki-1): An antigen (glycoprotein) associated with activation of hematopoietic cells of B, T, and monocyte origin.
CD71 (Ber-T9): An activation antigen that defines the transferrin receptor. It is expressed on activated T cells, bone marrow blasts, normal histiocytes, and intermediate- and higher-grade lymphomas, the Reed–Sternberg and Hodgkin cells of Hodgkin lymphoma, and other nonhematopoietic rapidly growing neoplasms.
HLA-DR: Expressed normally on B lymphocytes; however, HLADR is negative on quiescent T lymphocytes. It is expressed on activated T lymphocytes.
Ki-67 (MIB-1): The Ki-67 antibody detects a nuclear-associated antigen that is expressed by proliferating, but not resting cells. Ki67 staining correlates with morphologic grade, whereby a higher number of staining cells are associated with a poor survival.
panels on paraffin-embedded tissue T cell:
CD2
CD3
CD43
CD5
CD7
CD62L
CD8
CD4
CD30
TdT
Beta F1
NFATc1
TOX
CD52: clone, YTH34.5 or Campath-1G; concentration, 1:500
reverse transcriptase in situ hybridization assays
Epstein–Barr virus-associated latent small nuclear RNA (EBER): EBER-1 and EBER-2, present in both the productive and various forms of latent EBV infection. We employ EBER rather than LMP-1 since EBER is present in both the latent and lytic phases of infection while LMP-1 is typically not present in the lytic stage. EBER-1 and EBER-2 are present in much higher copy numbers than LMP-1, potentially providing us with higher sensitivity than testing LMP-1 protein.
Viral thymidine kinase (vTK assay): EBV thymidine kinase detected with the probes 5′-GAACCCGCATGCTCTCCTT-3′ and 5′-TCTGGATGATGCCCAAGACA-3′, respectively, detects lytic infection.
HHV8: Detection of HHV8 RNA is accomplished using primers specific for the T0.7 viral message, which is expressed in latent and active infection.
Fluorescent
in-situ hybridization (FISh)
MYC amplification and translocation, and trisomy 8: For MYC amplification, a ratio of the total number of MYC signals to the total number of CEP8 signals, in at least 60 interphase nuclei with nonoverlapping nuclei in the tumor cells, is determined. Cells with no signals or with signals of only one color are disregarded. Tumor cells displaying at least two centromeric chromosome 8 signals and multiple MYC signals, with a MYC/CEP8 ratio ≥2, are considered consistent with amplification of the MYC gene. Overamplification of C-MYC is not associated with any particular hematologic malignancy, but would only be expected in those with a more aggressive course and would not be a feature of a benign lymphoid cell population. Tumor cells displaying multiple centromeric chromosome 8 signals and an approximately equal number of MYC signals with a somewhat random distribution of both probe signals are considered polysomy 8.
ALK-1 breakapart probe: The LSI ALK (anaplastic lymphoma kinase) dual color, breakapart rearrangement probe contains two differently labeled probes on opposite sides of the breakpoint of the ALK gene. This region is involved in the vast majority of breakpoints for known 2p23 rearrangements that occur in t(2;5) and its variants. The translocation (2;5)(p23;q35) is identified in approximately 50% of cases of anaplastic large cell lymphoma (noncutaneous). The absence of the translocation (2;5)(p23;q35) does not exclude the diagnosis of anaplastic large cell lymphoma and in primary cutaneous anaplastic large cell lymphoma it is primarily not seen.
Interferon regulatory factor 4-breakapart dual color probes: Translocations involving the multiple myeloma oncogene-1/interferon regulatory factor-4 (IRF4) locus on 6p25 in primary cutaneous anaplastic large cell lymphoma and a subset of lymphomatoid papulosis
cases. The 5′ IRF4 CTD-2308G5 probe is labelled with Cyanine3 (R for red) and the 3′ IRF4 RP11-164H16 probe with SpectrumGreen (G for green). After hybridization of 5′ and 3′ IRF4 probes, the normal diploid pattern is one of two fusion signals (2F); a chromosomal break point at the vicinity of IRF4 is associated with 1F-1R-1G pattern (1F-1 split), defining a translocation in this area of the genome.
MYC breakapart probe: The LSI MYC dual color, breakapart rearrangement probe is a mixture of two probes that hybridize to opposite sides of the region located 3′ of MYC. This region is involved in the vast majority of breakpoints for t(8;22)(q24;q11) and t(2;8) (p11;q24). Translocation involving the CMYC gene can be expected to occur in the vast majority (>90%) of Burkitt’s lymphoma and atypical Burkitt’s lymphoma.
MYC IgH fusion probe: The LSI IGH/MYC, CEP 8 tricolor, dual-fusion translocation probe is designed to detect the juxtaposition of immunoglobulin heavy chain (IGH) locus and MYC gene region sequences. The IGH probe contains sequences homologous to essentially the entire IGH locus, as well as sequences extending about 300 kb beyond the 3′ end of the IGH locus. The large MYC probe extends approximately 400 kb upstream of MYC and about 350 kb 3′ beyond MYC. A cell harboring the reciprocal t(8;14) with the 8q24 breakpoint well within the MYC probe target is expected to produce a pattern of one orange, one green, two orange/ green fusions, and two aqua signals. Translocation involving the C-MYC gene can be expected to occur in the vast majority (>90%) of Burkitt’s lymphoma and atypical Burkitt’s lymphoma.
Bcl-2 IgH fusion probe: The LSI IGH/bcl-2 dual-color, dual-fusion translocation probe (Vysis) is designed to detect the juxtaposition of immunoglobulin heavy chain (IGH) locus and bcl gene sequences. It is detected in most lymphomas harboring a t(14;18).
Cyclin D1 IgH fusion probe: The LSI IGH/CCND1 dual-color, dual-fusion XT translocation probe (Vysis) is designed to detect the juxtaposition of immunoglobulin heavy chain (IGH) locus and CCND1 gene sequences. It will detect most t(11;14)-bearing cells and is therefore seen in the majority of mantle cell lymphomas.
MALT1 breakapart probe: The LSI MALT1 dual-color, breakapart rearrangement probe consists of a mixture two FISH DNA probes. The first probe, a 460 kb probe labeled in SpectrumOrange™, flanks the 5′ side of the MALT1 gene. The second probe, a 660 kb probe labeled in SpectrumGreen™, flanks the 3′ side of the MALT1 gene. It will detect cells with t(18q21) and/or aneuploidy of chromosome 18. Translocation involving the MALT1 gene can be expected to occur in approximately 25–50% of extranodal marginal zone lymphomas, but is quite uncommon in nodal-based marginal zone lymphoma and primary cutaneous marginal zone lymphoma.
MALT1 IgH fusion probe: The LSI IGH/MALT1 dual-color, dualfusion translocation probe is composed of a mixture of a 1.5 Mb SpectrumGreen™ labeled IGH probe and a 670 kb SpectrumOrange™ labeled MALT1 probe. The IGH probe contains sequences homologous to essentially the entire IGH locus, as well as sequences extending about 300 kb beyond the 3′ end of the IGH locus. The LSI MALT1 probe contains sequences that extend from a point telomeric to the D18S531 locus, through the MALT1 and HAK genes, and end proximally at a point centromeric to the HAK locus. This probe is useful in identifying the IGH/MALT1 t(14;18)(q32;q21) translocation.
API2 MALT1 fusion probe: The LSI API2/MALT1 dual-color, dual-fusion translocation probe is composed of a mixture of a SpectrumGreen™ labeled IGH probe and a SpectrumOrange™ labeled MALT1 probe. This probe is useful in identifying the API2/MALT1 t(11;18)(q21;q21) translocation. It will detect cells with a t(11;18) (q21;q21) translocation.
Chapter 2
the therapy of Cutaneous t Cell Lymphoma
Benjamin h. Kaffenberger, Mark a. Bechtel, and pierluigi porcu
Introduction
Mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma (CTCL), generally is characterized by an indolent presentation and by a low probability of progression. Patients with limited patches or plaques (Stage IA) have a <10% risk of developing progressive disease and have median survival similar to that of age-matched controls without the disease (Kim et al., 1996). In a subset of patients with Stage IB-IIA, however, CTCL may progress to more extensive disease and in a small minority of cases it may present de novo with tumors, erythroderma, and peripheral blood or visceral involvement (Kim et al., 2003). In these circumstances CTCL is associated with a significant risk of disease-related mortality and shorter survival (Lu et al., 2001; Kim et al., 2003; Vonderheid and Bernengo, 2003; Tancrede-Bohin et al., 2004). Sézary syndrome (SS), a rare and unique primary leukemic form of CTCL, presents with erythroderma and peripheral blood lymphocytosis (Vonderheid and Bernengo, 2003). Prognosis for these patients is poor, with 5-year survival at best 30% (Tables 2.1 and 2.2).
In the absence of molecular biomarkers, there are currently no broadly applicable molecular tools for risk stratification in CTCL. Twist 1, a transcription factor that inhibits p53 and C-MyCinduced apoptosis, correlates with later stages of CTCL and Sézary syndrome, however it has not been validated as a risk factor of progression (Goswami et al., 2012). Advanced clinical stage, older age, elevated lactate dehydrogenase (LDH) levels, and peripheral blood eosinophilia are all associated with poor prognosis (TancredeBohin et al., 2004). Histopathologically, the presence of over 25% of
Table 2.1 CTCL therapies
Skin-directed
Topical corticosteroids
Topical nitrogen mustards: mechlorethamine and carmustine (BCNU)
Topical retinoids: bexarotene
Topical imiquimod
PUVA (psoralen plus ultraviolet A) phototherapy
Narrowband UVB phototherapy
Electron beam radiotherapy
Photodynamic therapy
Systemic
Extracorporeal photophoresis
Retinoids: bexarotene and acitretin
Interferon-α
Histone deacetylase inhibitors (HDACi): romidepsin and vorinostat
Pralatrexate
Brentuximab vedotin
Alemtuzumab
Denileukin diftitox (not currently available)
Chemotherapy: Gemcitabine and doxorubicin
Hematopoietic stem cell transplantation
Table 2.2 Therapy by stage of mycosis fungoides
Stage Therapy
Stage IA-IB
Stage IIA
Stage IIB and above
Topical steroids, topical mechlorethamine, topical BCNU, narrowband UVB, PUVA, total skin electron beam radiotherapy, topical or systemic bexarotene
Same as above
Interferon-α, bexarotene, PUVA, combinations of topical and systemic treatment, HDACi –romidepsin or vorinostat, brentuximab vedotin, pralatrexate, chemotherapy, alemtuzumab, extracorporeal photophoresis (ECP), hematopoietic stem cell transplantation
large, atypical, CD30-positive cells, indicates large cell transformation, which has a poor prognosis, and is further worsened by older age or tumor stage disease (Diamandidou et al., 1998). Conversely, the expression of the chemokine receptors CCR4, CXCR3, and CXCR4 on malignant T cells is generally restricted to earlier stage lesions and loss of these markers with increased levels of CCR7, a lymph node homing receptor, is observed in patients with tumor stage disease and lymphadenopathy (Lu et al., 2001; Kallinich et al., 2003). Finally, loss of epidermotropism in advanced forms of MF, such as SS, is a common finding, and skin biopsies in these patients may not show any tumor cells within the epidermis.
A number of defects of adaptive and innate immunity can be observed during the clinical progression of CTCL (Kim et al., 2005).
Chronic and excessive production of Th2 cytokines, such as IL-4, IL-5, and IL-10, is believed to be an important mechanism by which malignant T cells circumvent antitumor responses. Gradual loss of Th1 cytokines (IL-12 and IFN-γ), CD8-positive cytotoxic T cells (CTL), and natural killer (NK) cells are observed in advanced-stage CTCL (yoo et al., 2001; French et al., 2005). Low absolute numbers of peripheral blood or skin CD8-positive T cells, measured by flow cytometry or immunohistochemistry, have been reported to be an accurate predictor of survival (Abeni et al., 2005), and therapy with retinoids has led to increased CD8-positive T cells in responders.
Diagnostic work-up and staging procedures
Staging evaluation of CTCL patients should include a comprehensive physical examination, a complete blood count, a comprehensive metabolic panel, and the quantification of circulating malignant T cells by flow cytometry. In patients presenting with typical MF, bone marrow aspirate and biopsy, contrast-enhanced computed tomography (CT) scan, and whole body positron emission
Cutaneous Lymphoid Proliferations:
of Lymphocytic Infiltrates of the Skin, Second Edition. Cynthia M. Magro, A. Neil Crowson and Martin C. Mihm.
tomography (PET) scanning should only be performed if diffuse lymphadenopathy, unexplained peripheral blood findings, or clinical signs or symptoms of visceral involvement are present. In patients with non-MF-type CTCL, a comprehensive staging and diagnostic work-up to verify that the disease is limited to the skin should always be performed. The role of T cell receptor (TCR) rearrangement analysis in the routine staging evaluation of CTCL remains to be established. Detection of clonal TCR-β rearrangements in typical skin lesions of patients with suspected CTCL for diagnosis and discovery of identical clones in the peripheral blood, lymph nodes, or bone marrow is unequivocal evidence of extracutaneous extension. However, in the absence of histopathological or immunophenotypical evidence of disease, the clinical significance, and therefore the treatment implications, of finding TCR-β rearrangements in the bone marrow, lymph nodes, or peripheral blood of CTCL patients is the subject of continuous debate (Delfau-Larue et al., 1998; Assaf et al., 2005).
CtCL therapies
The therapeutic choices for the treatment of CTCL depend on the stage of the disease and the general health and age of the patient. Although the therapies may be effective in controlling the disease, they have not been shown to prolong life. Because CTCL can involve skin, blood, bone marrow, and lymph nodes, proper staging is critical in the management of this disease. The treatment of CTCL can be divided into skin-directed and systemic therapies (see Table 2.1). Therapeutic choices may be challenging due to limited randomized clinical trials for CTCL. Therapeutic approaches using combination therapies may have synergistic benefits and may reduce toxicity of the single agents. Although combined modalities may increase disease-free survival, they do not change overall survival (Duvic et al., 2003). The primary goals of therapy are to improve the quality of life, induce diseasefree remission, and prolong life. Localized skin-directed therapies can be very successful in managing localized disease. Patients with more widespread disease need total skin-directed therapy or systemic therapy.
Skin-directed therapies
Topical corticosteroids
The use of topical corticosteroids is often effective in controlling early stage mycosis fungoides (see Table 2.2). Limited patch and thin plaque disease responds most consistently. A complete response in 60% of patients was reported in early-stage disease with topical corticosteroids (Zacheim et al., 1988). Generally, cost-effective topical steroids include hydrocortisone 2.5% cream/ointment for facial and intertriginous sites, triamcinolone 0.1% cream/ointment for the majority of the body, and fluocinonide 0.05% cream/ointment for recalcitrant areas and small body surface areas. The benefit of utilizing topical steroids over topical immune modulators, retinoids, and nitrogen mustards lies in their inexpensive nature. The side effects of topical steroids include cutaneous atrophy, telangiectasias, striae, and, rarely, suppression of the pituitary adrenal axis with systemic absorption in high concentrations and high body surface area applications.
Topical chemotherapy
Topical nitrogen mustards, such as mechlorethamine and carmustine (BCNU) intercalate between DNA strands and inhibit DNA
replication. They have been demonstrated to be effective in the management of early stages of mycosis fungoides.
For over 30 years mechlorethamine had to be compounded for use in the treatment of mycosis fungoides. Studies of the compounded forms demonstrated complete remissions in approximately 60–80% of patients with early patch and plaque-stage disease. Most longlasting remissions occurred in patients with patch or plaque-stage mycosis fungoides without palpable lymphadenopathy (Vonderheid et al., 1989).
Compounding requires preparing 10 mg of mechlorethamine in 50–60 mL of water and applying with a brush to the entire skin surface, except eyelids, lips, and rectal and vaginal orifices. This is repeated daily for 6–12 months. If patients develop a hypersensitivity reaction manifested as cutaneous erythema and pruritus, the treatment can be briefly interrupted. After the hypersensitivity reaction subsides, a more dilute solution (10 mg in 500 mL of water) can be initiated as tolerated. The concentration can be slowly increased over time. After a complete remission is achieved, the treatments can be gradually tapered, but no tapering schedule has demonstrated superior clinical efficacy. Mechlorethamine can also be applied in a mineral oil (Aquaphor®) base and may be less irritating. However, as the gel form is now FDA approved, compound pharmacists will no longer be able to dispense the old form without a good reason (e.g. the patient found the gel base to be too drying or irritating).
Topical carmustine (BCNU) has proved effective in early stage mycosis fungoides. It can be applied in a stock solution of BCNU in alcohol or prepared in an ointment base with white petrolatum. A complete response was documented in 86% of patients with Stage IA and 47% with Stage IB disease. The median time for a complete response was 11.5 weeks. Approximately 18% of patients were relapse-free at five years (Zackheim et al., 1990). The cutaneous side effects of topical carmustine include skin tenderness, erythema, and hyperpigmentation. Many patients develop increased telangiectasias and thus this should be avoided on the face. Allergic contact dermatitis and primary contact irritation develop in a similar way to topical mechlorethamine. Myelosuppression can develop with topical use and should be carefully monitored with complete blood counts.
Mechlorethamine is now FDA approved for MF in a 0.02% gel, preventing the need to compound these agents. This was after a large multicenter trial showed noninferiority to the compounded ointment (Lessin et al., 2013). However, this study did demonstrate the gel form possessed a more rapid onset of action. Unfortunately about 20% of patients in both arms withdrew due to skin irritation. Mechlorethamine can also be used as adjunctive therapy with other modalities, but should be avoided when using ultraviolet light therapy due to an increased risk of skin cancer.
Topical retinoids
Bexarotene gel, a synthetic retinoid X agonist, has been approved for the treatment of mycosis fungoides. The retinoid receptors (RAR and RXR) are members of a family of transcription factors belonging to the nuclear hormone receptor family. The nuclear hormone receptor family also includes thyroxine receptor, vitamin D receptor, and peroxisome proliferator-activated receptor. Bexarotene induces an RAR–RXR heterodimer complex that activates gene promoter regions encoding transcription factors, structural proteins, and cell receptors. This results in transcriptional modulation of cell function and differentiation, growth inhibition, and apoptosis.
The safety and efficacy of topical bexarotene gel was examined in an open-label Phase III study of 50 patients with refractory or persistent early-stage CTCL. A partial response was noted in 54% of patients and a complete response in 10% (Heald et al., 2003). In a Phase I/II study of bexarotene gel, 61 patients with early stage CTCL were treated. Partial responses were noted in 42% of patients and complete responses in 21% (Brenenman et al., 2002).
Bexarotene gel is initiated at a dose of 1% topical gel every other day and gradually increased in frequency to four times a day at 1–2 week intervals. The drug can cause a retinoid erythema and irritant dermatitis. It may take several weeks to note a clinical response.
The immunologic effects of bexarotene are related to a decreased expression of TH2 cytokines and decreased monocyte macrophage inflammatory mediators. There is a decrease in abnormal keratinocyte proliferation and induction of apoptosis of abnormal T cells.
Topical imiquimod
Imiquimod 5% cream has been demonstrated to be helpful in limited patch and plaque-stage mycosis fungoides (Suchin et al., 2002; Deeths et al., 2005). Imiquimod is a topical immunomodulator via toll-like receptor 7 activation, which induces localized interferon production. In one study, three of six patients with Stage IA to IIB mycosis fungoides were reported to have histologic clearing and significant clinical improvement with imiquimod 5% cream applied three times per week for 12 weeks. Patients responded best to a concomitant therapy with PUVA, systemic retinoids, or systemic interferon (Deeths et al., 2005).
Phototherapy
Psoralen plus UVA exposure (PUVA) has become a standard therapy for patch and plaque-stage CTCL (Herrmann et al., 1995). A long-term study of CTCL and PUVA at Northwestern showed 66 of 104 patients with clinical Stages IA to IIA achieving complete remission, which in many cases was long term. Disease-free survival rates at 5 years and 10 years for Stage IA disease were 56% and 30%, respectively. For Stage IB/IIA, the 5 year disease-free survival was 74% and 10 year disease-free survival was 50%. However, the 15-year survival rate for Stage IA was 82% and for Stage IB/IIA was 58% (Querfeld et al., 2005). Once the patient achieves a maximal response to treatment, maintenance therapy is usually required to prevent relapse. Persistent or recurrent lesions particularly favor UV-shielded sites. PUVA therapy can be combined with oral bexarotene, interferon-α, and radiotherapy.
The side effects of PUVA include erythema, burn injury, increased frequency of melanoma and nonmelanoma skin cancers, and cataracts. Protection of the eyes with ultraviolet light-absorbing goggles is necessary for 24 hours following ingestion of oral psoralens.
Narrowband UVB is an effective treatment for patch and thin plaque mycosis fungoides. It is less effective than PUVA for thick plaque disease. In one study, patients with IA and IB mycosis fungoides achieved complete remission in 81% of cases (Dierderen et al., 2003).
Narrowband UVB does not require photoprotection of eyes, unlike PUVA, and may have a lower risk of secondary skin cancers. It may be less effective than PUVA for thicker plaques. Narrowband UVB also requires multiple treatments per week initially, which make compliance an important issue.
Radiation therapy
Total skin electron beam (TSEB) therapy is often very effective for patients with early stages of mycosis fungoides. An energy of
4–6 MeV is delivered to the skin with penetration limited to the upper dermis. The limited penetration of electrons spares deeper parenchymal tissues, bone marrow, and the gastrointestinal tract. Approximately 3600 cGy are delivered in fractionated doses over 8–12 weeks. Separate exposures may be necessary for difficult to reach areas, including the palms, soles, axillae, perineum, and scalp. Complete remissions were reported in 98% of patients with Stage T1 disease, 71% with Stage T2 disease, and 36% with Stage T3 disease. Approximately 50% of patients with Stage T1 and 20% of with Stage T2 were disease-free at 10 years (Hoppe et al., 1979). Relapse is highest in patients with tumors, lymphadenopathy, and visceral involvement. With the highly fractionated approach, patients can receive a second course of electron beam therapy to reinduce remission. However, unlike phototherapy, it cannot be used continuously and relapses will occur. Hence, this method is often used for clearance before attempting a hematopoietic stem cell transplant. Otherwise, after stopping the TSEB, other skin-directed therapies should be added for maintenance.
The toxic side effects of TSEB therapy may be acute or chronic. Acute side effects include erythema, edema, bullae, and desquamation. Adnexal structures that may be affected include the hair follicles and sweat glands. Patients consequently may experience hypohidrosis, alopecia, and shedding of nails. Chronic side effects include hyperpigmentation, telangiectasias, cutaneous atrophy, xerosis, and anhidrosis. As the total dose of radiation increases, so does the risk for squamous cell carcinoma.
Cutaneous lymphomas are often very radiosensitive and local radiotherapy can be utilized for patients with individual tumors or thick plaques. It may be used in combination with other modalities, such as PUVA. Approximately 800–3000 cGy can be delivered in fractionated doses.
Photodynamic therapy
Photodynamic therapy (PDT) has been reported to be beneficial in the treatment of mycosis fungoides. PDT involves photosensitization with 5-aminolevulinic acid and irradiation with a noncoherent light source. Activation of the photosensitizer leads to the formation of oxygen radicals, which are highly reactive and cytotoxic. PDT has the potential to inhibit the proliferation of malignant T lymphocytes (Ammann and Hunziker, 1995). This may be a useful treatment modality in patients who have reached a partial remission with more conventional therapies. The therapy can be directed to therapyresistant lesions, but is not an option for large surface areas.
Goals of therapy in advanced-stage CtCL
It is now established that by the time CTCL has advanced to tumor lesions, the malignant T cells have acquired multiple molecular abnormalities in the pathways that regulate normal cell growth and survival. These defects are responsible for the high resistance to drug-induced apoptosis displayed by malignant T cells and the brief and partial responses typically seen with conventional therapy in CTCL. Intensive cytotoxic chemotherapy, as used in other forms of lymphoma and leukemia, may actually shorten survival in some patients by virtue of its myelosuppressive effect and its depression of T-cell-mediated immunity and provides few durable responses. Thus, the focus of systemic therapy in CTCL has gradually evolved from conventional cytotoxic chemotherapy to agents that induce T cell apoptosis by inhibiting survival pathways or targeting surface markers, and enhance the patient’s ability to mount an immune response, while avoiding neutropenia.
extracorporeal photopheresis (eCp)
ECP is a form of systemic therapy, originally developed by Edelson et al. (1987), that is FDA approved for the treatment of SS. In ECP, patients ingest oral 8-methoxypsoralen (8-MOP); then peripheral blood lymphocytes are collected by leukopheresis, exposed ex vivo to ultraviolet A (UVA) light, and reinfused back into the patient. ECP is initially performed on two consecutive days once a month until maximal clearing has occurred. This is followed by an additional 6 months of monthly therapy and then gradually tapered to 2 month intervals and eventually discontinued. In the initial cohort reported by Edelson et al. (1987), the response rate in refractory CTCL (most of whom had SS) was 73% and the median survival was 60 months. The side effects of ECP consist mostly of nausea and vomiting associated with oral 8-MOP. Newer formulations use liquid 8-MOP that is administered directly into the photopheresis machine, so patients do not have to ingest the psoralen. The proposed mechanism of action of ECP is twofold: (1) UVA irradiation directly kills malignant T cells sensitized by 8-MOP, and (2) the reinfused leukophoresis product stimulates a selective immune response against the malignant cells. Further studies have retrospectively compared the outcomes of patients treated with or without ECP, generally finding similar survivals. Overall, ECP appears to be effective only in a subset of patients with advanced-stage CTCL, specifically SS with near normal CD8+ T cell counts, and a relatively short duration of advanced disease. However, these patients are also more likely to respond to other immune-modifying therapies and the value of ECP vis-à-vis other treatment modalities has not been prospectively tested in clinical trials (Richardson et al., 2003). Finally, due to the highly encouraging responses seen with alemtuzumab in SS and due to the fact that ECP requires special equipment, a significant time commitment, and lack of ubiquity at medical centers, it is conceivable that monoclonal antibodies will become more frequently used for this indication than ECP.
Interferons
The most active single agent in the treatment of CTCL is IFN-α. Initial studies in heavily pretreated patients with CTCL, with doses ranging from 36 million international units (MIU) per day to 50 MIU three times a week, showed objective response rates of 45–65%, with complete response rates of 10–30% and response duration of 4.5–5.5 months (Olsen and Bunn, 1995). In patients with all stages of CTCL who had not received prior systemic therapy, the overall response rate was as high as 80%. Enthusiasm for the high response rate with IFN-α has been tempered by its significant toxicity profile. Adverse effects to IFN-α include malaise, fever, chills, myalgias, depression, anorexia, and weight loss. Although none of these events are dose-limiting in isolation, their cumulative burden is commonly severe and this fact often leads to substantial dose modification or cessation of therapy. Frequent laboratory abnormalities observed during therapy with IFN-α include dose-related and reversible leukopenia and thrombocytopenia, abnormal liver function tests, and, occasionally, hypothyroidism. There is clearly a dose–response effect with IFN-α in CTCL. The current treatment approach with IFN-α is to start at 3 MIU per day and increase as tolerated, usually to a maximum of approximately 15 MIU per day. Lower doses of IFN-α have been successfully combined with other treatment modalities with excellent results. At Northwestern University, a study combining the use of IFN-α (3–12 MIU three times a week) with PUVA in patients with all stages of MF/SS demon-
strated an overall response rate of 88%, with a complete response rate of 62% and median response duration of 28 months, establishing this combination as one of the most active therapies in CTCL (Kuzel et al., 1995).
In addition to IFN-α, there is evidence that systemic or topical recombinant IFN-γ may have significant potential for the treatment of CTCL, and that it may be better tolerated than IFN-α (Kaplan et al., 1990; Dummer et al., 2004). In addition to enhancing the CTL function, IFN-γ suppresses excess Th2 cytokine production, enhances CD40 expression, and primes abnormal dendritic cells for IL-12 production, particularly in response to CD40 ligation.
retinoids
Retinoids are vitamin A derivatives and have been shown to have antiproliferative and differentiating effects in many hematological malignancies, including CTCL (Zhang and Duvic, 2003). Response rates ranging from 44% to 68% have been reported with isotretinoin (13-cis-retinoic acid) in patients with Stage I–II disease (Fitzpatrick and Mellette 1986; Kessler et al., 1987). Due to its teratogenic properties with regard to teen pregnancies, this medication is highly regulated, which limits its use, including the requirement for monthly physician evaluations and updates of the iPledge registry. The most common toxicities include drying of the skin and mucous membranes, conjunctivitis, fatigue, arthralgias, mental status changes, headaches, and elevated triglyceride levels. More recently, bexarotene, a retinoid X receptor (RXR) selective agonist, was shown to have substantial clinical efficacy in both topical and oral formulations in patients with all stages of CTCL and was approved in 1999 by the FDA for this indication (Duvic et al., 2001). In the pivotal trial, 94 patients with CTCL received oral bexarotene at doses ranging from 300 to 650 mg/m2/day. At a dose of 300 mg/m2/day, the response rate observed was 45–55%, depending on stage, with a median time to response of 180 days and a median duration of response of 10 months. There was a nonstatistically significant trend toward a dose–response relationship, with greater complete response rate (13%), shorter time to response (59 days), and longer duration of response (13 months) at doses higher than 300 mg/m2. However, dose-limiting toxicity (hypertriglyceridemia) prevented continuation of therapy at higher doses (650 mg/m2/day) of bexarotene and the FDA-approved dosage is 300 mg/m2/day. Adverse events included hypertriglyceridemia, hypercholesterolemia, central hypothyroidism, and leukopenia. The activity of bexarotene in the non-MF/SS-type of CTCL has not been investigated in clinical trials but responses in CD30-positive primary cutaneous ALCL and panniculitis-like T cell lymphoma have been reported.
Bexarotene exerts a number of biological effects that may sensitize malignant T cells to other therapies. At clinically relevant concentrations, bexarotene upregulates both the p55 and p75 subunits of the interleukin-2 (IL-2) receptor in vitro, thus enhancing the susceptibility of T cell leukemia cells to denileukin diftitox (see “Immunotoxins”) by five- to tenfold. To determine whether this effect could be reproduced in vivo, Foss et al. (2005) completed a small Phase I study of bexarotene (75–300 mg/day) and denileukin diftitox (18 μg/kg per day × 3 days every 21 days) in 14 patients with relapsed or refractory CTCL. Overall response was 67%, with four complete responses. Modulation of IL-2R expression on CTCL cells was observed at or above a bexarotene dose of 150 mg/day. Leukopenia and lymphopenia were observed in a small number of patients, without clinical evidence of opportunistic infections. Thus,
the combination of denileukin diftitox and bexarotene was well tolerated and even low doses (150 mg/day) of bexarotene were capable of upregulating CD25 expression (Foss et al., 2005).
Acitretin is the other systemic retinoid that is still available in the United States. It is not RXR specific nor FDA approved for this indication, unlike bexarotene, but it has shown an overall response of nearly 60% in small patient numbers (Cheeley et al., 2013) and may be an option if a patient cannot tolerate the side effects of bexarotene.
Immunotoxins
Denileukin diftitox (DAB389IL-2, Ontak, Ligand Pharmaceuticals), a genetically engineered fusion protein combining the enzymatically active domain of diphtheria toxin and the full-length sequence of interleukin-2 (IL-2), efficiently targets cells expressing the highaffinity IL-2 receptor subunits that are highly expressed on the malignant T cells in the majority of cases of CTCL. After binding to the IL-2 receptor, denileukin diftitox undergoes endocytosis followed by release of the diphtheria toxin, which results in arrest of protein synthesis and, ultimately, apoptosis of T cells. The initial Phase I study with denileukin diftitox in 35 patients consisted of five daily infusions every 3 weeks for up to six cycles at escalating doses, yielding an overall response rate of 37% with 14% complete responses. Adverse effects of DAB389IL-2 included fever, chills, hypotension, nausea, vomiting, and elevated transaminases. In the following pivotal Phase III trial in heavily pretreated patients, two different doses of denileukin diftitox (9 μg/kg and 18 μg/kg) were studied (Olsen et al., 2001). The overall response rate was 29.5%,with 10% complete responses and no difference between the two dose levels. A mild, self-limited form of vascular leak syndrome (VLS) is frequently observed after the first cycle of denileukin diftitox, with lower extremity edema, hypoalbuminemia, and elevated serum creatinine. The frequency and intensity of VLS can be significantly reduced by premedication with dexamethasone (4–8 mg orally) without compromising the efficacy of therapy. Currently, denileukin difititox is approved for use only in CD25-positive CTCL, as defined by positive staining by immunohistochemistry (IHC) on frozen or paraffin embedded tissue. However, the levels of CD25 expression sufficient to confer sensitivity to denileukin diftitox are likely to be much below the IHC threshold of detection, as suggested by the numerous responses reported in CD25-negative CTCL in early clinical trials.
The major mechanism of action of denileukin diftitox in CTCL is believed to be direct killing of malignant T cells. However, part of its activity may also be immunologically mediated through the elimination of CD4/CD25-positive regulatory T cells (Tregs), a normal subset of T cells with constitutive suppressive activity (Dannull et al., 2005). Thus, denileukin diftitox, by depleting Treg cells, may conceivably enhance normal residual antitumor responses in CTCL patients. Unfortunately, at the current time, this therapy is no longer available in the United States.
Monoclonal antibodies
A number of humanized monoclonal antibodies (mAbs) against surface antigens expressed by normal and malignant T cells are available for clinical use. Alemtuzumab (Campath-1H) is a humanized IgG1 mAb directed against CD52, a glycosylated peptide antigen expressed on most malignant B and T cells. Alemtuzumab was developed primarily for the treatment of patients with B cell
chronic lymphocytic leukemia (B-CLL); however, it also showed strong activity in T cell leukemias and lymphomas. Lundin et al. (2003) prospectively studied alemtuzumab in 22 patients with advanced CTCL. The overall response rate was 55%, with 32% complete responses, and median duration of response approaching 12 months. Impressively, Sézary cells were cleared from the blood in 86% of the patients. Cytomegalovirus (CMV) reactivation occurred in 18% of patients, but without clinical respiratory or gastrointestinal syndromes. Three patients had more significant infectious complications, such as generalized herpes simplex, aspergillosis, and mycobacterial pneumonia. Larger studies have also corroborated an impressive benefit, particularly in patients with SS (de Masson et al., 2014). Thus, alemtuzumab shows promising activity, particularly in patients with erythroderma and SS, but its use requires aggressive prophylactic therapy and surveillance for opportunistic infections. It appears that alemtuzumab may become one of the drugs of choice for SS patients and further studies are in progress.
In addition to alemtuzumab, a number of additional mAbs targeting T cell markers such as CD2 and CD4 have been recently studied. Preliminary results from a Phase I clinical trials of siplizumab (MEDI-507), a humanized anti-CD2 mAb, in patients with various types of T cell lymphoma have been presented. Typical infusional adverse reactions occurred with the first dose and asymptomatic CMV reactivation was seen. Treatment resulted in partial remission or stabilization of disease in several patients. However, cases of EBV-induced lymphoproliferative disorders, some of them fatal, were observed and further development of the drug in CTCL was stopped.
Another mAb in early clinical development is zanolimumab (HuMaxCD4) (Hagberg et al., 2005). Phase II data with this antibody in 47 CTCL patients relapsing after bexarotene showed response rates ranging from 22% to 75%, including some complete responses, depending on the dose (280, 560, or 980 mg) and the stage of disease (Kim et al., 2007). More than 80% of the responses were reached within 8 weeks and median response duration was more than 10 months. Nine patients with SS were treated on this study at all dose levels, four of which obtained minor responses and one a partial response. The observed responses in these patients were generally short-lived and depletion of CD4 T cells was limited, suggesting that this antibody may not be as effective as alemtuzumab in SS. Although promising, the maker discontinued the Phase III trial due to slow patient recruitment and limited market potential and it has not been further pursued.
The mAb that has the greatest potential for reaching the CTCL market in the US at this time is mogamulizumab, a humanized glyco-engineered (defucosylated) antibody targeting the CC chemokine receptor 4 (CCR4). The defucosylated Fc region enhances antibody-dependent cellular cytotoxicity (ADCC). CCR4 is mainly expressed on normal Tregs and type 2 helper T cells (TH2). CCR4 is highly expressed on adult T-cell leukemia/lymphoma (ATLL) cells and mogamulizumab is currently approved in Japan for the treatment of ATLL. In addition, mogamulizumab depletes CCR4+ Tregs, potentially evoking antitumor immune responses (Ni et al., 2015). Clinical trials with mogamulizumab have demonstrated clinical efficacy and tolerability for the treatment of relapsed/refractory T-cell lymphomas, including CTCL. A Phase I/II multicenter, doseescalation study of mogamulizumab was conducted in relapsed patients with PTCL and CTCL (Duvic et al., 2010). Forty heavily pretreated patients received at least four doses of KW-0761 at 0.1 mg/kg (n = 3), 0.3 mg/kg (n = 3) and 1 mg/kg (n = 34); 38 patients (23 with MF; 15 with SS) were evaluable for efficacy. The objective
response rate (ORR) was 39%, with two patients achieving CR (5%), and 13 achieving partial response (PR; 34%). With SS patients, 12 of 15 (80%) had a response in the blood, including seven (47%) CRs. SD was seen in 19 patients (50%), whilst PD was observed in four patients (11%). As in the first study, the maximum tolerated dose was not reached. A Phase III randomized clinical trial comparing mogamulizumab with the histone deacetylase inhibitor vorinostat in patients with relapsed/refractory CTCL is currently recruiting (ClinicalTrials.gov Identifier: NCT01728805).
histone deacetylase inhibitors (hDaCi)
The accessibility of nuclear chromatin to transcription factors and RNA polymerase is regulated in part by the balance of acetylation of nucleosomal histones, which is the result of the opposing activities of histone deacetylases (HDACs) and histone acetyl transferases (HATs). Deacetylation-induced silencing of tumor suppressor genes, via HDAC, has been observed in a variety of human cancers. Restoration of gene expression through inhibition of histone deacetylation is a novel form of therapy that selectively induces growth arrest, differentiation, and apoptosis in malignant cells. Several families of HDACi have been characterized. Compounds such as suberoylanilide hydroxamic acid and depsipeptide, now vorinostat and romidepsin, respectively, have entered the clinical arena and shown promise in the treatment of T cell lymphoma (Piekarz et al., 2004). In a Phase II study, 27 patients with advanced CTCL were treated with vorinostat given as a 4-hour infusion on days 1, 8, and 15 of a 28-day cycle (Piekarz et al., 2005). Three patients with Sézary syndrome achieved a complete response and five other patients had partial responses for an overall response rate of 30%. The median duration of response is 18 months (range 6–48). A larger study of heavily pretreated patients in advanced stages showed overall response rates of 34% and 38% in a subgroup analysis of only advanced disease (Piekarz et al., 2009, Whittaker et al., 2010). Overall, romidepsin was well tolerated, with dose-related, but modest nausea, fatigue, neutropenia, and thrombocytopenia. However, a number of severe cardiac adverse events have occurred in clinical trials with this agent and monitoring during infusions is necessary. Vorinostat is another potent HDACi, which is prescribed orally at 400 mg daily and is FDA approved for advanced primary CTCL. Of 37 patients, 10 treated with various oral dosing regimens of vorinostat achieved a partial response, including a decrease of lymphadenopathy (Duvic et al., 2005). In addition, nearly half of patients had relief from CTCL-associated pruritus (Duvic et al., 2007). Unfortunately, it had to be discontinued in several patients because of fatigue, rash, anemia, neuropathy, thrombocytopenia, and weight loss. The follow-up Phase IIb study of heavily pretreated, advanced cases of CTCL demonstrated an overall response of 29.7%, with high frequencies of the same adverse events, but fortunately low-grade (Olsen et al., 2007). Overall, HDACi appear to be very active in CTCL and are effective second-line agents.
antibody drug conjugates (aDC)
Brentuximab vedotin is an antibody drug cytotoxic conjugate made up of an antibody to CD30 Fab′ domains conjugated with monomethyl auristatin, an antimitotic agent receptor. It is dosed at 1.8 mg/kg every 21 days and is FDA approved in the treatment of refractory Hodgkin lymphoma and systemic anaplastic large
cell lymphoma. As far as CTCLis concerned, efficacy has been reported, including complete response, in patients with primary cutaneous anaplastic large cell lymphoma (Kaffenberger et al., 2013). As CD30 positivity is a marker of transformed mycosis fungoides, results have also been seen in these cases, including four patients with Stage IV disease, one patient with complete resolution, and two patients with a partial response (Mehra et al., 2015), which allowed stem cell transplantation in one patient. Interestingly, another report details a complete response and partial response in two separate patients with transformed disease, despite variable CD30-positive expression indicating testing for CD30 expression may be insensitive and that there may be an effect in nontransformed MF (Saintes et al., 2015). A Phase II study is ongoing to determine the efficacy in cases of cutaneous lymphomas. Neuropathy is an important adverse event, but there are also a growing number of reports of progressive multifocal leukencephalopathy (Carson et al., 2014).
Studies are ongoing for the use of brentuximab vedotin in cutaneous T-cell lymphomas; the initial reports appear very promising and although not FDA approved, the agent may be available for use in refractory and compassionate cases.
Cytotoxic chemotherapy
Systemic cytotoxic chemotherapy has generally been used for palliation of CTCL patients with relapsed or refractory disease or for patients with advanced disease at presentation. Methotrexate, glucocorticoids, cyclophosphamide, cisplatin, etoposide, bleomycin, doxorubicin, vincristine, and vinblastine have all been used as single agents or in combination, with complete response rates up to 30%, but very short remission durations and significant toxicity, mostly due to myelosuppression (Rosen and Foss, 1995). Prednisone alone induces partial remissions in approximately 50% of patients and pulse dexamethasone is effective in obtaining symptomatic relief in patients with severe SS. However, flares are the rule once the dose is tapered and chronic steroid use is associated with significant morbidity.
The purine analogs, such as 2′-deoxycoformycin (pentostatin), 2-chlorodeoxyadenosine (2-CDA), and fludarabine have profound effects on normal and malignant T cells. Response rates in CTCL have been higher with 2-deoxycoformycin (54–100%) than with the latter two compounds, but no prospective comparative study has been performed (Grever et al., 1983; Cummings et al., 1991; Kuzel et al., 1996; Quaglino et al., 2004). Major toxicities of these drugs are neurotoxicity and prolonged immunosuppression, leading to opportunistic infections.
Trimetrexate (TMTX) is a lipophilic antifolate that enters cells by passive diffusion and achieves intracellular concentrations 10–100 times greater than those of MTX, thus bypassing possible mechanisms of resistance. In addition, TMTX is not polyglutamated and is not retained in cells for prolonged periods, possibly accounting for its relatively low hepatotoxicity. Based on the known clinical activity of MTX in CTCL, a Phase II study of TMTX (200 mg/m2 IV every 14 days up to 12 doses) in patients with relapsed CTCL was recently completed, showing a 45% response rate in heavily pretreated patients and tolerable toxicity (Sarris et al., 2002). Although this drug is probably significantly more active, the intravenous route of administration makes it unlikely to replace MTX. Pralatrexate, another antifolate is now FDA approved for the treatment of peripheral T cell lymphoma. It is an infusion given weekly, typically best-tolerated for MF patients at 15 mg/m2 for 3–4 weeks
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noodige zouden behoeven te doen, en [170]in geen geval iemand van dien kant, Ella uitgezonderd, in hun huis zouden behoeven te ontvangen. En Carel mocht geen cent van Wiechens geld aannemen. Dat geld, door woeker of misdrijf verkregen, kon toch nooit geld zijn waar zegen op rustte.
Carel had alles beloofd en toegegeven. Als hij Ella maar had, was hij tevreden; meer begeerde hij niet.
Wiechen was verrast.
Aan deze mogelijkheid had hij nu het allerminst gedacht! Dat een meisje, dat men laat studeeren, het in haar hoofd krijgt, om vlak vóór het eindexamen van het Gymnasium, zich te willen engageeren, vond hij iets onbehoorlijks. Dat beetje, dat volgens hem voor een getrouwde vrouw noodig is, had men goedkooper kunnen leeren. Nu was het weggegooid geld, en daar hield hij niet van.
Hij wilde niets beslissen, zoo dadelijk, en begon, om tijd te winnen, den jongen man de vragen te stellen, die hij meende dat bij een gelegenheid als deze gebruikelijk waren, te weten naar Carels toekomst en de financieele positie zijner ouders.
En Carel, die van beide niets dan goeds kon zeggen, vond dit een gunstig teeken, en antwoordde zoolang hij wat te zeggen had, terwijl Wiechen luisterde en nu en dan korte vragen deed. De macht der gewoonte toonde zich ook hier weer. Het was langzamerhand of het een te sluiten geldleening betrof. Werktuigelijk begon Wiechen de gegevens, die hem Carel gaf, met potlood op een voor hem liggend stuk papier op te teekenen.
„Ik zal informatie nemen,” zeide hij opeens. „Wanneer moet het geld er zijn?”
„Geld, meneer?” riep Carel verbaasd uit. „Daar heb ik in de allerlaatste plaats aan gedacht, of liever in het geheel niet.” [171]
„O neen,” zeide Wiechen, zich herstellende. „Ik was een oogenblik absent. Maar nu we er toch over spreken, konden we dat ook behandelen. U verwacht natuurlijk, dat Ella wat meekrijgt. Misschien moet ik daarover met uw papa spreken …”
„Neen meneer. Vader is … te oud,” redde zich Carel, die begreep dat nu het moeielijke punt kwam. „Hij mag nergens in gemengd worden, te wille van zijn gezondheid. Hij geeft zijn toestemming, ziedaar alles. En het zou hem en mij aangenaam zijn, als over geld niet werd gesproken. Met mijn tractement en de toelage, die mijn vader zal geven, is te dien aanzien gezorgd.”
„Zoo,” deed Wiechen langzaam. „Nu, we zullen zien. Ik had andere plannen met haar, en wil eerst weten, of het haar ernst is die op te geven. En dan, moet ik haar moeder erin kennen.”
„Wanneer mag ik dan mijn antwoord verwachten?”
„Morgenavond om dezen tijd. Is dat goed? Iets vroeger kan ook, maar niet vóór zessen. Als de zaak doorgaat, zullen we toch nog een en ander te bespreken hebben …”
„Ik zal om half zeven hier zijn,” beloofde Carel, opstaande.
Het was een vervelend geval, vond Wiechen. Hij wilde dat Ella onbezorgd zou zijn in de toekomst, en dat bood zich nu aan, al was het niet op de manier, die hij gedacht had. Want dat ooit iemand, laat staan in Holland, om zijn dochter zou komen, had hij niet verwacht. Maar hij had een tweede bedoeling. Zoodra zij klaar was, wilde hij haar aan zijn practijk verbinden, door haar gedekt zijn. En dát ontging hem op deze wijze.
„Ella!” riep hij, en het antwoord kwam van zóó dicht bij, dat hij besefte, dat zij zich in de buurt van het kantoor had opgehouden.
„Ga eens zitten,” zeide hij, toen zij binnenkwam, de [172]sporen dragend van te hebben gehuild. „Je weet wat er geweest is.”
En op haar toestemmend knikje, deelde hij haar mee, niet geheel te hebben willen weigeren, maar dat zijn inzicht in de zaak was, dat men het moest laten afhangen van het eindexamen. Dat begon nu over enkele dagen. Slaagde zij, dan bleek het, dat zij voldoende studeerkracht had, om ook verder te komen. Men zou zoolang het antwoord kunnen uitstellen.
„Dat is niet noodig, pa,” zeide Ella. „In dat geval kunnen we nu evengoed beslissen.”
„Ben je zóó zeker, dat je er komt?”
„Neen,” zeide Ella, zacht doch beslist. „Ik zou het diploma graag hebben, en zal er mijn best voor doen. Maar als ik dan Carel niet hebben mag … dan laat ik mij expres zakken.”
Vloekend sprong hij op en schudde haar doorelkaar.
Zij bood niet den minsten tegenstand.
„Zoo zal het ook op het examen gaan,” zeide zij apatisch. „Geen behoorlijk antwoord zal men uit mij krijgen.”
Hij liet haar los. Ja, dat was wel zijn dochter! Zoo was ook hij geweest, toen hij indertijd zijn militair pensioen wilde halen.
„Gévédé! Trouw dan voor mijn part. Maar doe ook je examen.”
„Dat beloof ik u,” riep Ella uit, hem om den hals vallend. „U meende het toch ook niet. wel? U wou maar eens zien of het mij ernst was, nietwaar?”
„Al goed,” bromde hij.
Zij keek op de klok.
„Mag ik het nog even aan moeder gaan zeggen?” vroeg zij. „En als ik thuis kom, zal ik gauw een briefje aan mevrouw schrijven. De post zal haar adres toch wel weten.” [173]
[Inhoud]
HOOFDSTUK XVI.
De voorspelling van Dr. Arnolds was uitgekomen. Zijn zoon en Viehof werden vrijgesproken. Beiden waren in vrijheid gesteld, en men had hun de effecten moeten teruggeven, die destijds in beslag genomen waren. Die waren nu op de gewone wijze in Engeland verkocht, waartoe Boom daarheen was gegaan. Zijzelf hadden dat niet aangedurfd, maar op Boom, die van onbesproken reputatie was, kon geen verdenking vallen. En hij had dit te eer gedaan, daar dit de eenige manier was om aan het hem rechtmatig toekomend deel van den buit te komen. Heel eerlijk zond men ook Hervau zijn deel, zich herinnerende, dat deze bad verteld nog meer zaken te hebben, waaraan geld te verdienen was.
Er was een tweede reden, waarom Boom de geschiktste persoon voor dit karreweitje was geweest. Ieder wist, dat hij naar het buitenland ging om te trouwen. Dat had men in alle couranten onder de huwelijksaankondigingen kunnen lezen. Het was niet meer dan natuurlijk, dat hij een poosje vóór den dag der voltrekking er al heen ging. Zulke dingen duurden altijd wat, en reeds was men verbaasd geweest over de snelheid waarmee aan de formaliteiten, die de wetten van beide landen voorschrijven, was voldaan, dank zij den door Arnolds aanbevolen advocaat, de eenige die [174]over zijn werk niet tevreden was, daar hij buiten een klein voorschot, geen honorarium ontvangen had.
Maar in elk geval, de effecten waren verkocht, en Boom was getrouwd teruggekomen. De dankbaarheid der familie zijner vrouw was ook meegevallen, zoodat hij thans netjes ingericht woonde. Het eenige wat tegenviel, was dat hij in alles op de vingers gekeken werd door zijn vrouw, en ongenadig onder de plak zat.
Geen opstaan tegen den middag, geen vertoef in koffiehuizen des nachts; slechts bij hoogst enkele gelegenheden mocht hij alleen uit.
Ook in geldzaken hield zij de teugels strak. Niets mocht bij doen zonder haar geraadpleegd te hebben, en alle accepten of ander papier nam zij hem af, om het zelf te bewaren, en er hem tegen de vervaldagen mee uit te sturen. Zijn zakgeld was afgepast.
Enkele dagen na zijn thuiskomst had Boom de anderen op een glas wijn genoodigd, daar Arnolds had medegedeeld een zaak te weten. En daar mevrouw Boom van oordeel was geweest, dat men die zaak evengoed, ja veiliger thuis kon bespreken, dan in Café Central, was men thans bijeen.
De zaak was een renpaard te koopen, en op de Hollandsche banen te laten loopen. Het paard was op de rennen nog nagenoeg onbekend, en had feitelijk pas een paar maal in het buitenland geloopen tot proef.
„Zit Hervau erin?” vroeg Boom, en op toestemmend knikken, ging hij voort: „Dan is de zaak veilig. Hij kent dat. Maar hoeveel kost die grap?”
„Voor ons een kleinigheid,” zeide Viehof. „Enkel stallen en oppassen, gedurende een week of wat. Hervau stuurt het beest over, en wij betalen hier. Maar hoofdzaak is, dat wij hem moeielijk op onzen naam kunnen laten loopen. Arnolds of ik tenminste niet. Wil jij dat doen?” [175]
„Neen” zeide Boom. „Voor mij is het ook niets. Maar ik weet een eerzaam winkelier, die wel eens een beetje gokt, en die het wel voornaam zal vinden. Ik sta voor hem in.”
„Duur?”
„Neen. We mogen hem er geen te groote zaak van voorspiegelen; anders wantrouwt hij het. Zoo voor de aardigheid, om ook eens als eigenaar te figureeren en nog een kleinigheid toe te verdienen, waarvoor hij zoo goed als niets waagt. Want we moeten hem iets mee laten betalen; anders is het te mooi.”
„Best,” zeide Arnolds. „Zorg jij dan voor je winkelier en de inschrijving, en wij doen de rest. Ik zal dadelijk telegrafeeren, dan kan de knol op de volgende courses te Woestduin meeloopen.”
„Maar tegen een onbekend paard zijn geen pari’s te krijgen,” meende Boom.
„O, hij moet de eerste keer ook niet winnen. Een keer of drie moet hij meeloopen. Tweemaal zóó verliezen, dat er een hooge côte te maken is, en de derde maal in een verkoopsren winnen. Zie je, dat is de manier, om geld te verdienen, en tegelijk de lastpost van het paard kwijt te zijn. Wacht, ik zal mijn telegram klaarmaken.”
En hij zette zich tot schrijven.
Ruim drie weken later was het drietal vereenigd te Woestduin. Het was de groote dag, waarop Miss Kate, hun paard winnen zou. In de volle zekerheid van hun slagen, hadden Arnolds en Viehof zoowel bij de bookmakers als aan den totalisator hun geld geplaatst, het over verschillenden verdeelende, om geen argwaan te wekken. Boom had gewacht. Het was zijn systeem, om eerst te zien wat vóórging, en zoo laat mogelijk te zetten, in elk geval niet eer men wist of alle paarden meeliepen. Eindelijk besloot [176]hij het voorbeeld der anderen te volgen, en verwijderde zich van hen. Eensklaps werd hij zacht aan zijn mouw getrokken. Het was een gewezen jockey, die thans het baantje van adviseur uitoefende, en tips gaf. Boom had hem vroeger eens uit den nood geholpen, en daar werkelijk soms
goeden raad voor ontvangen, de enkele malen, dat hij naar de courses ging.
„Speelt u Walkyrie” raadde hij.
„Ik was van plan Miss Kate te spelen,” zeide Boom.
„Dat dacht ik wel,” zeide de ander. „Ik heb u zien praten met meneer Viehof. Die zit in een combinatie.”
„Hoe drommel weet je dat?” riep Boom uit.
„Ik moet wel alles weten, tenminste als er geknoeid wordt. De jockey is omgekocht, geloof u me gerust. Miss Kate wint ditmaal niet. De volgende maal.”
Boom begreep, dat daar iets achter moest zitten. Het was een verkoopsren, en men moest verkoopen, omdat men het financieel niet langer houden kon. Won dus Miss Kate niet, dan was men paard en inzetten kwijt.
„Weet je het heelemaal zeker?” vroeg hij.
„Heelemaal zeker,” antwoordde de ander. „Maar ik zou niemand anders dan u gewaarschuwd hebben. U verklikt me niet?”
„Dankje wel,” zeide Boom. „Kom straks eens bij mij.”
„Neen, meneer, dan zoude men misschien iets merken. Ik kom morgen avond even bij u thuis.”
Boom volgde den gegeven raad, maar met kloppend hart. Hoewel de ex-jockey nooit met zekerheid tevoren kon zeggen, welk paard zou winnen, als elk zijn best deed, kon hij toch daartegenover wel
veilig afraden een paard te spelen, als hij van knoeierij de lucht had. En daarop was hij uitgeslapen.
Het leek een spannende strijd. Na eerst in een klomp te [177]hebben geloopen, drongen reeds na de eerste hindernis twee paarden vooruit. En ofschoon de achterblijvers hoe langer hoe meer distantie kregen, die twee bleven als aan elkaar hangen.
„Hij houdt Miss Kate in,” zeide Arnolds zacht tegen de anderen. „Dat is zeer verstandig. Dan kan hij haar krachten voor het laatste vlakke eind bewaren. Het gaat goed.”
Maar een oogenblik later uitte hij onwillekeurig een kreet.
Bij den sprong over de laatste hindernis was Miss Kate teveel op de voorhand neergekomen, en had door den schok zijn vaart verloren. Het scheen zelfs een oogenblik of hij vallen zou, zoo wankelde de rijder. Zich onmiddellijk weer herstellende schoot het paard echter weer vooruit, zichbaar den voorsprong inhalende, dien Walkyrie door dat korte oponthoud gekregen had. Maar telaat. Met een neuslengte was Miss Kate geslagen!
Hij had mooi in de angst gezeten. Toen hij het geval bij de laatste hindernis zag, begreep hij dat dit een gewilde misstap was, doch later twijfelde hij weer. En minder goed gezicht op de paarden hebbende, daar de tribune eenigzins schuin tegenover den winpaal stond, dacht hij een oogenblik dat Miss Kate tòch weer voor was gekomen. Doch neen, en weldra weerklonk het geroep: allright! waarmee de overwinning van Walkyrie een feit werd.
Arnolds en Viehof stonden met bleeke gezichten te kijken.
„Ik moet eerst wat drinken,” zeide de laatste, waarmee de ander instemde.
„En ik moet wat versche lucht blijven inademen,” zeide Boom. „Is me dat een toestand! Ik kom straks ook aan het buffet.”
Zoodra zij daarin verdwenen waren, ging hij zijn winst [178]realiseeren. Eerst bij de bookmakers, waar het van uit het buffet meer in het gezicht kon loopen, en toen bij den totalisator. Daarna vervoegde hij zich bij de anderen.
„Geen prettig bericht voor Hervau,” begon Boom.
„Voor ons is het erger,” antwoordde Arnolds. „Hoeveel heb jij gezet?”
„Duizend pop.”
„’t Is jammer. Ik wou dat die vervloekte knol de beenen gebroken had. De jockey heeft z’n best gedaan. Het scheelde nog maar een haartje.”
„Gévédé!” liet Viehof los.
„Kom, er is niets aan te doen,” meende Boom. „Laat ons opwandelen, en aan het hôtel nog iets pakken. Die champagne begint me tegen te staan.”
En zij zetten zich in beweging, langzaam opwandelend naar de door Boom voorgestelde plaats, om van daaruit de komst van den trein af te wachten.
Thuisgekomen dacht Boom zijn vrouw te verheugen met al het geld, dat hij gewonnen had. Doch dat was een misrekening. Wel nam zij het hem af, maar diende hem een ongezouten standje toe. Ten eerste omdat hij de duizend gulden, waarmee hij gespeeld bad, niet
had afgedragen, en voorgewend, dat hij die de vorige week niet had kunnen innen. En voorts, omdat hij die gewaagd had, en zeker kwijt zou geweest zijn, als niet toevallig die jockey hem had ingelicht. Enfin, het was gebeurd, en nu gelukkig afgeloopen; maar hij moest het niet weer wagen. Neen, nooit meer. Want ze hadden genoeg, om met voorzichtig uitzetten van te leven, en gingen gaandeweg zelfs vooruit. Er was dus geen enkele reden om van die gewaagde dingen te doen.
„Het was niet gewaagd,” wierp hij tegen. „Geknoei kan niemand verhelpen.” [179]
„Spelen is altijd gewaagd,” besliste zij. „En ik wil het niet meer hebben, hoor je!”
Maar later bleek zij toch niet zoo kwaad, daar zij Boom honderd gulden meegaf, om Arnolds en Viehof, als die thans zonder geld zaten, elk wat te geven. En hij mocht dien avond alleen naar Central, om het hun te brengen. Want sedert zij getrouwd was, kon ze moeielijk meer daarheen gaan, met de kans de maîtresses der anderen te ontmoeten.
Zoo slenterde Boom na den eten naar het bewuste Café, hoewel hij nauwelijks verwachtte de anderen daar te vinden. Zeer verwonderd was hij dus hen te zien niet alleen, maar blijkbaar hadden zij met Fifi en Leda goed gedineerd, en waren in vroolijke stemming.
„Goed bericht!” riep hem Arnolds toe, zoodra hij hem bespeurde.
„Een telegram van Hervau. Hij seint het paard nog aan te houden, en een volgenden keer te laten loopen. Hij zal stalgeld vergoeden en dubbel onze inzetten, die er vandaag bij ingeschoten zijn.”
„Begrijp je dat?” voegde Viehof erbij.
„Nogal eenvoudig,” zeide Boom. „Hij heeft tegen zijn eigen paard gespeeld, en zelf gezet op Walkyrie.”
„Bliksems! Wat heeft hij daaraan?”
„Om tweemaal te winnen. Door onze hooge inzetten leek het, of Miss Kate nu liep om te winnen. Dat depreciëerde de côte van Walkyrie, waardoor hij op dat paard zettende, meer won. Den volgenden keer denkt men, dat het wéér zoo gaan zal, en slaan we onzen slag met Miss Kate. En dan is het uit.”
„Waarom liet dan de jockey Miss Kate zoo gevaarlijk ophalen?”
„Om een disqualificatie te ontgaan. Daar zijn ze bier tegenwoordig zoo bijster vlug mee. We hebben hem dadelijk [180]teruggeseind hoeveel stalgeld en inzet is. Voor jou ook.”
„Je hebt er toch wat opgelegd?”
„Neen, zeker niet. Onder elkaar eerlijk, hè?”
„Natuurlijk,” zeide Boom, even kleurende. „Ik maakte ook maar gekheid. Kom, dat is een goed bericht, en ik geef er een dozijntje oesters op, bij Hoek.”
Dat denkbeeld vond de noodige instemming, en men maakte zich langzamerhand op, om de uitnoodiging te volgen.
Toen Boom dien avond naar huis ging, aarzelde hij, of hij zijn vrouw mededeeling zou doen van het extra fortuintje, dat hem te beurt viel. Zij had gelijk, het was wagen geweest. Maar het zou zonde zijn de volgende keer, als Miss Kate werkelijk won, niet weer mee te kunnen doen. Heelemaal niets zeggen, durfde hij ook niet. Hij moest toch eenigen uitleg geven, waarom hij de honderd gulden niet aan de anderen had behoeven te geven, maar er van had gesoupeerd met
hen. Als het nu niet zoo laat was geworden, en hij kon ergens het meerdere geld bergen, was het wat anders. Ook deed zich de moeielijkheid op, zoodra hij de twee mille van Hervau in zijn zak had, te maken, dat Helene er de lucht niet van kreeg. Zij ledigde zijn zakken, en borstelde dan zijn goed af. Inslikken kon hij het toch niet! Ha, hij wist iets! Hij kon haar wijsmaken, dat Hervau hem duizend gulden restitueerde, in de meening, dat hij die verspeeld had, en de andere duizend gulden om voor gezamenlijke rekening den volgenden keer te spelen. Zoo was elk tevreden, en de volgende maal zou hij de helft afdragen, en de andere helft quasi aan Hervau moeten zenden. Tegen dien tijd zou hij er wel een geschikt plekje voor vinden, waar zij niet bij kon.
Het was lastig, zoo’n vrouw, die overal haar neus instak. En toch kon hij niet buiten haar. Reeds hadden, toen hij [181]pas thuis was, een paar oude schuldeischers geprobeerd hem aan het kleed te komen, maar ze moesten onverrichterzake afdeinzen, omdat alles van háár was. Hij had zich toen gehaast dit zooveel mogelijk bekend te maken, om geen herhaling te krijgen. En bij slot van rekening zorgde zij toch goed voor hem, en was het in zijn eigen belang. Alleen, lastig was het.
Reeds den volgenden avond kwam de remise van Hervau. Dat was wat men noemde coulant! Men deelde, en gaf den winkelier te wiens name het paard liep, de voorgeschoten stalgelden terug. En deze, die al bang geworden was, dat men hem erin had laten loopen, en met een deurwaarder in overleg was getreden het paard in beslag te nemen, verheugde zich in den goeden afloop.
„Ik wist wel, dat ik u kon vertrouwen, meneer Boom,” zeide hij; „maar het loopt toch in de papieren. Ik heb er zelf vierhonderd gulden op gezet en verloren. Ik zal de volgende maal mijn inzet moeten
verdubbelen, en dan willen we er het beste van hopen. In elk geval weet ik, dat ik een renpaard heb gehad.”
„Maak u maar niet ongerust,” zeide Boom. „Het was een toeval, dat het paard ditmaal heeft verloren. Aanstaanden Zondag zal het wel beter gaan.”
En deze voorspelling kwam uit.
Mooier dan ditmaal Miss Kate liep, had nooit eenig paard geloopen. Hands down werd de ren gewonnen, Vroolijker dan de vorige maal gingen nu ook Arnolds en Viehof naar huis.
„Ik ga er een beetje vandoor,” zeide de eerste. „Ga je mee?”
„Waarheen?”
„Forest, Namur, of zoo. En als we geluk verder.” [182]
„Top,” antwoordde Viehof. „Gauw wat inpakken, en met den avondtrein tot Brussel?”
„Best!”
„Wat zijn jelui toch dom,” meende Boom. „Nu heb je wat geld, en kunt met een beetje beleid er van leven, en af en toe nog eens een slagje slaan. Doe zooals ik, en blijf hier. ’k Weet van tevoren, dat jelui sans le sou terugkomt.”
„Ik heb toch eens tachtigduizend francs gewonnen,” merkte Arnolds op.
„Ja, en die heb je er in een maand doorgelapt,” zeide Boom. „Op die manier heb je er niet veel aan.”
„Het komt er niet opaan,” zeide Arnolds. „We hebben dan tenminste plezier gehad. Wat heb je hier?”
„En,” vond Viehof, „als we terugkomen, en het is op, dan zoeken we maar wéér wat op.”
„Ik houd me aanbevolen,” verklaarde Boom.
Zij deden zooals afgesproken, en spoedden zich naar hun kamers, om hun koffertje te pakken, en de meisjes wat geld te geven. Maar deze lieten zich daarmee niet afschepen, en het gevolg was, dat zij zoolang aandrongen tot ze mee mochten.
Even voorbij de grens hadden zij hun gewone bezigheid hervat, als ze reisden, te weten Arnolds en Viehof kaart te spelen, de meisjes te kijken en te babbelen, toen zich in de deur van den coupe, die ze voor den rook hadden opengelaten, de figuur van Wiechen vertoonde.
„Zoo, heertjes,” zeide hij, „éérste klasse en de dames mee?”
„Kom bij ons zitten,” noodigde Arnolds. „Waar ga jij heen?”
„Naar Parijs,” zeide Wiechen. „Neen, ik kom niet binnen. Ik heb tweede klasse, en als een conducteur komt.…”
Zij drongen aan, maar hij bleef weigeren, vertellende van een koopje dat hij eens gehad had door dat te doen. [183]En bovendien was hij niet alleen. Een dame, die hij wegbracht.
„Blijf je lang?” vroeg Arnolds.
„Ik weet het nog niet. Eens kijken, of ik er iets van mijn gading vinden kan. Nu, we zien elkaar in de wachtkamer te Brussel wel. Of gaan jelui niet door?”
„Vannacht blijven we te Brussel. Bonjour!”
„Ik moet toch zien wie hij bij zich heeft,” verklaarde Arnolds, toen Wiechen verdwenen was. „Neen, één voor één, asjeblieft,” ging hij voort, ziende dat ook de anderen opstonden.
Hij bleef maar kort weg.
„Gordijntjes dicht,” vertelde hij. „Door de reet zag ik oen knap snoetje, maar onbekend.”
De overigen schenen het nu niet meer de moeite waard te vinden, en men ging door met spelen en praten. [184]
[Inhoud]
HOOFDSTUK XVII.
Ella Wiechen had haar eindexamen met goed gevolg afgelegd. Zoolang zij daarvoor werkte, had men het geraden geacht het engagement nog geheim te houden, teneinde haar geen te groote afleiding te geven. Nu zij er door was, wilde men haast maken met het huwelijk. Carel kon elken dag zijn orders krijgen, om naar Indië te vertrekken, en hoe vlugger alles afliep, hoe beter, naar de betrokken partijen voorkwam.
Zelfs Wiechen werkte mee. Want hij vreesde elken dag voor een beslag, en wilde zien vóór dien tijd weg te komen. Een paar kleine vonnisjes had hij afgedaan, doch een der groote hing er teveel in. Tot overmaat van ramp had de advocaat van van Vleuten, met hoop op een verstek, maar ineens gedagvaard, en daar Wiechen geen kosten meer wou maken, was de toeleg gelukt.
Van Marie van Groningen had Ella antwoord gekregen, waarbij deze haar hartelijk geluk wenschte. Als Ella trek had, haar eens op te zoeken, dan kon dit nu geschieden, daar zij bij haar oom en tante Slot was ingetrokken.
Gaarne gaf Ella nu aan die uitnoodiging gehoor, en nam ook dankbaar Marie’s aanbod aan, om haar met haar uitzet te helpen, zoodat zij geen onbruikbare dingen meenam.
Ten huize harer aanstaande schoonouders was zij in het [185]eerst met eenige koelheid ontvangen, doch ook die week weldra, en al heel spoedig was Ella maar zelden thuis te vinden.
Het begon er ook ongezellig te worden. Haar vader had, behalve op het kantoor, dat nog zijn gewone uiterlijk behield, overal ingepakt, en
veel de deur uitgezonden. Tot haar had hij gezegd, dat hij onmiddellijk na haar huwelijk naar Parijs zou vertrekken, en dus op de meubels na, alles wilde hebben opgeruimd en opgezonden. Zoo ging dan het een vóór, het ander na, de deur uit, en kreeg het eigenlijke huis den indruk of het voor een verkooping klaargemaakt was.
Op den Zaterdagmiddag, vóór den Zondag, waarop Wiechen naar Parijs spoorde, had hij Ella met een groot envelop naar Marie van Groningen gezonden, dat zij onmiddellijk moest overhandigen, en er haar bij gezegd, dat hij even heen en weer naar Parijs ging, en zij de gastvrijheid van Marie maar moest inroepen. Daarop had hij haar een hartelijken afscheidskus gegeven. Dit laatste herinnerde zich Ella pas later, toen Marie haar had verteld, wat er nader in den brief van haar vader stond.
Het was geen „even heen en weer”, maar een vertrek voor goed. En hij vroeg zijn vroegere vriendin, die toch zooveel van Ella hield, of zij haar bij zich wilde houden, en voor het verdere zorgen. Een acte van toestemming voor Ella’s huwelijk, was in den brief vervat. Die had hij doen opmaken, omdat, afgezien van zijn vertrek naar Parijs, hij bij de huwelijksvoltrekking niet tegenover die trotsche menschen wou staan, die niet eens zijn kennis wilden maken. Hij zou nog niet zoo spoedig gegaan zijn, als niet Maandag een beslag in zijn huis verwacht werd, en hij daar Ella niet in kon laten zitten. Hij verzocht Marie van dit alles aan Ella enkel het hoognoodige te willen meedeelen.
Ella schreide haar oogen rood, en zoo vond haar Carel, [186]die haar kwam afhalen, om bij zijn ouders te gaan eten. Hoewel hij hartelijk blij was, dat hij Wiechen, die hem tegenstond, niet zou ontmoeten voorshands, gevoelde hij toch groote deelneming met zijn aanstaand vrouwtje, en stelde haar voor haar komen voor heden af te gaan
